Factor VII polypeptides that are modified and uses thereof

ABSTRACT

Modified factor VII polypeptides and uses thereof are provided. Such modified FVII polypeptides include Factor VIIa and other forms of Factor VII. Among modified FVII polypeptides provided are those that have altered activities, typically altered procoagulant activity, including increased procoagulant activities. Hence, such modified polypeptides are therapeutics.

RELATED APPLICATIONS

Benefit of priority is claimed to U.S. Provisional Application Ser. No.61/124,021, to Edwin Madison and Christopher Thanos, entitled “FACTORVII POLYPEPTIDES THAT ARE MODIFIED AND USES THEREOF,” filed Apr. 11,2008.

This application is related to corresponding International ApplicationNo. PCT/US09/002,248 to Edwin Madison and Christopher Thanos, entitled“FACTOR VII POLYPEPTIDES THAT ARE MODIFIED AND USES THEREOF,” filed Apr.10, 2009, which also claims priority to U.S. Provisional ApplicationSer. No. 61/124,021.

The subject matter of the above-referenced applications is incorporatedby reference in its entirety.

The subject matter of U.S. application Ser. No. 12/082,662, to EdwinMadison, Christopher Thanos, Sandra Waugh Ruggles and Shaun Coughlin,entitled “MODIFIED FACTOR VII POLYPEPTIDES AND USES THEREOF,” filed Apr.11, 2008, and corresponding International Application No.PCT/US2008/04795 to Edwin Madison, Christopher Thanos, Sandra WaughRuggles and Shaun Coughlin, entitled “MODIFIED FACTOR VII POLYPEPTIDESAND USES THEREOF,” filed Apr. 11, 2008, also is incorporated byreference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT DISCS

An electronic version on compact disc (CD-R) of the Sequence Listing isfiled herewith in duplicate (labeled Copy #1 and Copy #2), the contentsof which are incorporated by reference in their entirety. Thecomputer-readable file on each of the aforementioned compact discs,created on Apr. 10, 2009, is identical, 1200 kilobytes in size, andtitled 4919SEQ.001.TXT.

FIELD OF THE INVENTION

Modified therapeutic proteins are provided. In particular modifiedFactor VII polypeptides, which includes Factor VIIa and other forms ofFactor VII, and uses thereof are provided.

BACKGROUND

Hemostasis is the complex physiological process that leads to thecessation of bleeding. Platelets, plasma proteins, and blood vessels andendothelial cells are the three components of this process that eachplay an important role in the events that immediately follow tissueinjury and which, under normal circumstances, results in the rapidformation of a clot. Central to this is the coagulation cascade, aseries of proteolytic events in which certain plasma proteins (orcoagulation factors) are sequentially activated in a “cascade” byanother previously activated coagulation factor, leading to the rapidgeneration of thrombin. The large quantities of thrombin produced inthis cascade then function to cleave fibrinogen into the fibrin peptidesthat are required for clot formation.

The coagulation factors circulate as inactive single-chain zymogens, andare activated by cleavage at one or more positions to generate atwo-chain activated form of the protein. Factor VII (FVII), a vitaminK-dependent plasma protein, initially circulates in the blood as azymogen. The FVII zymogen is activated by proteolytic cleavage at asingle site, Arg¹⁵²-Ile¹⁵³, resulting is a two-chain protease linked bya single disulphide bond (FVIIa). FVIIa binds its cofactor, tissuefactor (TF), to form a complex in which FVIIa can efficiently activatefactor X (FX) to FXa, thereby initiating the series of events thatresult in fibrin formation and hemostasis.

While normal hemostasis is achieved in most cases, defects in theprocess can lead to bleeding disorders in which the time taken for clotformation is prolonged. Such disorders can be congenital or acquired.For example, hemophilia A and B are inherited diseases characterized bydeficiencies in factor VIII (FVIII) and factor IX (FIX), respectively.Replacement therapy is the traditional treatment for hemophilia A and B,and involves intravenous administration of FVIII or FIX, either preparedfrom human plasma or as recombinant proteins. In many cases, however,patients develop antibodies (also known as inhibitors) against theinfused proteins, which reduces or negates the efficacy of thetreatment. Recombinant FVIIa (Novoseven® (Coagulation Factor VIIa(Recombinant))) has been approved for the treatment of hemophilia A or Bpatients that have inhibitors to FVIII or FIX, and also is used to stopbleeding episodes or prevent bleeding associated with trauma and/orsurgery. Recombinant FVIIa also has been approved for the treatment ofpatients with congenital FVII deficiency, and is increasingly beingutilized in off-label uses, such as the treatment of bleeding associatedwith other congenital or acquired bleeding disorders, trauma, andsurgery in non-hemophilic patients.

The use of recombinant FVIIa to promote clot formation underlines itsgrowing importance as a therapeutic agent. FVIIa therapy leavessignificant unmet medical need. For example, based on clinical trialdata, an average of 3 doses of FVIIa over a 6 hour or more time periodare required to manage acute bleeding episodes in hemophilia patients.More efficacious variants of FVIIa are needed to reduce theserequirements. Therefore, among the objects herein, it is an object toprovide modified FVII polypeptides that are designed to have improvedtherapeutic properties.

SUMMARY

Provided herein are modified Factor VII (FVII) polypeptides. Inparticular, provided herein are modified FVII polypeptides that exhibitprocoagulant activities. The FVII polypeptides are modified in primarysequence compared to an unmodified FVII polypeptide, and can includeamino acid insertions, deletions and replacements. Modified FVIIpolypeptides provided herein include FVII polypeptides that exhibitthose that have increased resistance to inhibitors such as antithrombinIII (AT-III) and tissue factor pathway inhibitor (TFPI), those that haveincreased resistance to the inhibitory effects of Zn²⁺, those that haveincreased catalytic activity in the presence and/or absence of TF, thosethat have improved pharmacokinetic properties, such as increasedhalf-life, those that have increased binding and/or affinity for theplatelet surface, those that have increased binding and/or affinity forserum albumin, and those that have increased binding and/or affinity forplatelet integrin α_(IIb)β₃. The modified FVII polypeptides can containany combination of modifications provided herein, whereby one or moreactivities or properties of the polypeptide are altered compared to anunmodified FVII polypeptide. Typically the modified FVII polypeptideretains procoagulant activity. Also provided herein are nucleic acidmolecules, vectors and cells that encode/express modified FVIIpolypeptides. Pharmaceutical compositions, articles of manufacture, kitsand methods of treatment also are provided herein. FVII polypeptidesinclude allelic and species variants and polypeptides and other variantsthat have modifications that affect other activities and/or properties.Also included are active fragments of the FVII polypeptides that includea modification provided herein. Exemplary of FVII polypeptides are thosethat include the sequence of amino acids set forth in SEQ ID NO:3, aswell as variants thereof having 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitytherewith.

Provided herein are modified factor VII (FVII) polypeptides that containa modification in a FVII polypeptide at position Q286 in a FVIIpolypeptide having a sequence of amino acids set forth in SEQ ID NO:3 orin corresponding residues in a FVII polypeptide. The modification can bean amino acid replacement, amino acid insertion(s) or deletion(s), orcombination thereof. In instances where the modification is an aminoacid replacement, replacement can be by a basic amino acid (e.g. Arg(R), Lys (K) and His (H)) or an amino acid selected from among Arg (R),Lys (K) His (H), Tyr (Y), Gly (G), Phe (F), Met (M), Ile (I), Leu (L),Val (V), Pro (P), Glu (E), Trp (W), Asp (D), and Cys (C). Exemplary ofsuch amino acid replacements include Q286R, Q286K, Q286H, Q286Y, Q286G,Q286F, Q286M, Q286I, Q286L, Q286V, Q286P, Q286E, Q286W, Q286D, andQ286C. Such modifications can be made in an unmodified FVII polypeptidecontaining a sequence set forth in any of SEQ ID NOS: 1-3, or an allelicor species variant thereof, or a variant having at least 60% sequenceidentity with the FVII of any of SEQ ID NOS: 1-3, or an active fragmentof a FVII polypeptide that comprises a sequence of amino acids set forthin any SEQ ID NOS: 1-3, or an allelic or species variant thereof, or avariant having at least 60% sequence identity with the FVII of any ofSEQ ID NOS: 1-3. For example, a modified FVII polypeptide can be anactive fragment that contains replacement at a position corresponding toposition Q286 in a FVII polypeptide.

In some examples, the modified FVII polypeptides provides herein containan amino acid replacement at a position corresponding to position 286 ina FVII polypeptide having the sequence of amino acids set forth in SEQID NO:3 or in a corresponding residue in a FVII polypeptide, wherein themodification is replacement at position 286 by a basic amino acid thatresults in a modified FVII polypeptide that exhibits increased coagulantactivity compared to the FVII polypeptide that does not have themodification at position 286. The basic amino acid can be selected fromamong Arg (R), Lys (K) and His (H). For example, a modified FVIIpolypeptide provided herein can contain a replacement of Gln (Q) withArg (R) at position 286.

In some examples, the modified FVII polypeptides have only the singlemodification at position 286. In other examples, the modified FVIIpolypeptides also contain one or more further modifications at anotherposition in the FVII polypeptide. The further modification can be anamino acid replacement, insertion or deletion. For example, the furthermodification can be an amino acid replacement at a positioncorresponding to a position selected from among A51, S52, P54, S60, Q66,Y68, K109, S119, A122, G124, T130, E132, V158, K161, A175, D196, K197,K199, R202, H216, S222, G237, T239, H257, Q286, L287, R290, A292, A294,E296, M298, L305, S314, G318, P321, K337, K341, Q366, H373, F374, E394,P395 and R396. Exemplary of such modifications include D196K, D196R,D196A, D196Y, D196F, D196W, D196L, D196I, K197Y, K197A, K197E, K197D,K197L, K197M, K197I, K197V, K197F, K197W, K199A, K199D, K199E, G237W,G237T, G237I, G237V, T239A, R290A, R290E, R290D, R290N, R290Q, R290K,R290M, R290V, K341E, K341R, K341Q, K341N, K341M, K341D, G237T238insA,G237T238insS, G237T238insV, G237T238insAS, G237T238insSA, D196K197insK,D196K197insR, D196K197insY, D196K197insW, D196K197insA, D196K197insM,K197I198insE, K197I198insY, K197I198insA, K197I198insS, T239S, T239N,T239Q, T239V, T239L, T239H, T239I, L287T, M298Q, P321K, P321E, P321Y,P321S, Q366D, Q366E, Q366N, Q366T, Q366S, Q366V, Q366I, Q366L, Q366M,H373D, H373E, H373S, H373L, H3731, H373F, H373A, K161S, K161A, K161V,H216S, H216A, H216K, H216R, S222A, S222K, S222V, S222N, S222E, S222D,H257A, H257S, Gla Swap FIX, {Gla Swap FIX/E40L}, {Gla Swap FIX/K43I},{Gla Swap FIX/Q44S}, {Gla Swap FIX/M19K}, {Gla SwapFIX/M19K/E40L/K43I/Q44S}, Gla Swap FX, Gla Swap Prot C, Gla Swap Prot S,Gla Swap Thrombin, S52A, S60A, E394N, P395A, R396S, R202S, A292N, A294S,G318N, A175S, K109N, A122N, G124S, A51N, T130N, E132S, S52N, P54S,S119N, L121S, T128N, P129A, Q66N, Y68S,S103S111delinsQRLMEDICLPRWGCLWEDDF, H115S126delinsQRLMEDICLPRWGCLWEDDF,T128P134delinsQRLMEDICLPRWGCLWEDDF, S103S111delinsIEDICLPRWGCLWE,H115S126delinsIEDICLPRWGCLWE, T128P134delinsIEDICLPRWGCLWE,S103S111delinsDICLPRWGCLWED, H115S126delinsDICLPRWGCLWED,T128P134delinsDICLPRWGCLWED, P406insIEDICLPRWGCLW,P406insGGGSIEDICLPRWGCLW, P406insDICLPRWGCLWED,P406insGGGSDICLPRWGCLWED, S103S111delinsSFGRGDIRNV,H115S126delinsSFGRGDIRNV, T127P134delinsSFGRGDIRNV, P406insCSFGRGDIRNVC,P406insGGGSCSFGRGDIRNVC, V158T, V158D, L287T, E296V, M298K and M298Q.

Exemplary of the modified FVII polypeptides provided herein are thosethat contain modifications Q286R/M298Q, Q286R/Gla Swap FIX, Q286R/H257A,Q286R/S222A, Q286R/S222A/H257A, Q286R/S222A/Gla Swap FIX,Q286R/H257A/Gla Swap FIX, Q286R/S222A/H257A/Gla Swap FIX,Q286R/M298Q/K341Q, Q286R/M298Q/K199E, Q286R/M298Q/Gla Swap FIX,Q286R/Q366V, Q286R/A292N/A294S/Q366V, A175S/Q286R/Q366V,S222A/Q286R/Q366V, H257S/Q286R, H257S/Q286R/Q366V,S222A/H257A/Q286R/Q366V, Q286R/H373A, S222A/H257A/Q286R/M158Q,Q286R/K341D, Q286R/Q366D, Q286R/Q366N, Q286R/M298Q/Q366D,Q286R/M298Q/Q366N, Q286R/H373F, Q286R/M298Q/H373F, {Gla SwapFIX/E40L}/Q286R/M298Q, {Gla Swap FIX/K43I}/Q286R/M298Q, {Gla SwapFIX/Q44S}/Q286R/M298Q, {Gla Swap FIX/M19K}/Q286R/M298Q, {Gla SwapFIX/M19K/E40L/K43V/Q44S}/Q286R/M298Q, T128N/P129A/Q286R,T128N/P129A/Q286R/M298Q, T128N/P129A/Q286R/H373F,V158D/Q286R/E296V/M298Q, Gla Swap FIX/T128N/P129A/S222A/Q286R, Gla SwapFIX/T128N/P129A/Q286R/M298Q, T128N/P129A/S222A/H257A/Q286R/M298Q,T128N/P129A/Q286R/M298Q/H373F, S52A/S60A/Q286R, Gla SwapFIX/S52A/S60A/S222A/Q286R, S52A/S60A/Q286R/M298Q, Gla SwapFIX/S52A/S60A/Q286R/M298Q, S52A/S60A/S222A/H257A/Q286R/M298Q,S52A/S60A/Q286R/H373F/, S52A/S60A/Q286R/M298Q/H373F, T239V/Q286R, GlaSwap FIX/S222A/T239V/Q286R, T239V/Q286R/M298Q,S222A/T239V/H257A/Q286R/M298Q, Gla Swap FIX/T239V/Q286R/M298Q,T239V/Q286R/H373F, T239V/Q286R/M298Q/H373F, T239I/Q286R, Gla SwapFIX/S222A/T239I/Q286R, T239I/Q286R/M298Q, S222A/T239I/H257A/Q286R/M298Q,Gla Swap FIX/T239I/Q286R/M298Q, T239/Q286R/H373F,T239V/Q286R/M298Q/H373F, Gla Swap FIX/S222A/Q286R/H373F, Gla SwapFIX/S222A/Q286R/M298Q, Gla Swap FIX/S222A/Q286R/M298Q/H373F,V158D/Q286R/E296V/M298Q/H373F, H257A/Q286R/M298Q, H257S/Q286R/M298Q, GlaSwap FIX/S222A/H257S/Q286R/, S222A/H257S/Q286R/M298Q,H257S/Q286R/M298Q/H373F, S222A/Q286R/M298Q/H373F, S222A/Q286R/M298Q,T128N/P129A/A175S/Q286R, A122N/G124S/A175S/Q286R, Gla SwapFIX/T128N/P129A/A175S/S222A/Q286R, Gla SwapFIX/A122N/G124S/A175S/S222A/Q286R, T128N/P129A/A175S/Q286R/M298Q,A122N/G124S/A175S/Q286R/M298Q, T128N/P129A/A175S/S222A/H257A/Q286R/M298Q, A122N/G124S/A175S/S222A/H257A/Q286R/M298Q,T128N/P129A/A175S/Q286R/M298Q/H373F,A122N/G124S/A175S/Q286R/M298Q/H373F, {Gla SwapFIX/K43I}/T128N/P129A/Q286R/M298Q, T128N/P129A/Q286R/M298Q/Q366N, {GlaSwap FIX/K43I}/Q286R/M298Q/Q366N, {Gla SwapFIX/K43I}/T128N/P129A/Q286R/M298Q/Q366N, V158D/Q286R/E296V/M298Q,T128N/P129A/Q286R/M298Q/Q366N/H373F, T239V/Q286R/M298Q/Q366N,T239I/Q286R/M298Q/Q366N, T128N/P129A/T239V/Q286R/M298Q,T128N/P129A/S222A/T239V/H257A/Q286R/M298Q,T128N/P129A/T239V/Q286R/M298Q/H373F, T128N/P29A/T239I/Q286R/M298Q orT128N/P129A/T239I/Q286R/M298Q/H373F.

Provided herein are modified FVII polypeptides containing two or moremodifications in a FVII polypeptide, allelic or species variant thereofor active fragments thereof. At least one of the modifications in suchpolypeptides is at a position corresponding to position Q286 in a FVIIpolypeptide having a sequence of amino acids set forth in SEQ ID NO:3 orin corresponding residues in a FVII polypeptide, providing that themodification at position Q286, alone or in combination with any othermodification, does not result in introduction of a new glycosylationsite compared to the unmodified FVII polypeptide. Such modifications canbe an amino acid replacement, insertion or deletion. For example, themodification at position Q286 can be a replacement by an amino acidselected from among Arg (R), Lys (K) His (H), Tyr (Y), Gly (G), Phe (F),Met (M), Ile (I), Leu (L), Val (V), Pro (P), Glu (E), Trp (W), Asp (D),and Cys (C). In some examples, the modification is Q286R. The one ormore other modifications can be selected from among D196K, D196R, D196A,D196Y, D196F, D196W, D196L, D196I, K197Y, K197A, K197E, K197D, K197L,K197M, K197I, K197V, K197F, K197W, K199A, K199D, K199E, G237W, G237T,G237I, G237V, T239A, R290A, R290E, R290D, R290N, R290Q, R290K, R290M,R290V, K341E, K341R, K341Q, K341N, K341M, K341D, G237T238insA,G237T238insS, G237T238insV, G237T238insAS, G237T238insSA, D196K197insK,D196K197insR, D196K197insY, D196K197insW, D196K197insA, D196K197insM,K197I198insE, K197I198insY, K197I198insA, K197I198insS, T239S, T239N,T239Q, T239V, T239L, T239H, T239I, L287T, P321K, P321E, P321Y, P321S,Q366D, Q366E, Q366N, Q366T, Q366S, Q366V, Q366I, Q366L, Q366M, H373D,H373E, H373S, H373F, H373A, K161S, K161A, K161V, H216S, H216A, H216K,H216R, S222A, S222K, S222V, S222N, S222E, S222D, H257A, H257S, Gla SwapFIX, {Gla Swap FIX/E40L}, {Gla Swap FIX/K43I}, {Gla Swap FIX/Q44S}, {GlaSwap FIX/M19K}, {Gla Swap FIX/M19K/E40L/K43I/Q44S}, Gla Swap FX, GlaSwap Prot C, Gla Swap Prot S, Gla Swap Thrombin, S52A, S60A, E394N,P395A, R396S, R202S, A292N, A294S, G318N, A175S, K109N, A122N, G124S,A51N, T130N, E132S, S52N, P54S, S119N, L121S, T128N, P129A, Q66N, Y68S,S103S111delinsQRLMEDICLPRWGCLWEDDF, H115S126delinsQRLMEDICLPRWGCLWEDDF,T128P134delinsQRLMEDICLPRWGCLWEDDF, S103S111delinsIEDICLPRWGCLWE,H115S126delinsIEDICLPRWGCLWE, T128P134delinsIEDICLPRWGCLWE,S103S111delinsDICLPRWGCLWED, H115S126delinsDICLPRWGCLWED,T128P134delinsDICLPRWGCLWED, P406insIEDICLPRWGCLW,P406insGGGSIEDICLPRWGCLW, P406insDICLPRWGCLWED,P406insGGGSDICLPRWGCLWED, S103S111delinsSFGRGDIRNV,H115S126delinsSFGRGDIRNV, T127P134delinsSFGRGDIRNV, P406insCSFGRGDIRNVC,P406insGGGSCSFGRGDIRNVC, V158T, V158D, L287T, E296V, M298K and M298Q.

In some examples, the modified FVII polypeptides contain a modificationat a position corresponding P54, Q66, L121, A122, P129 or E132 in a FVIIpolypeptide having a sequence of amino acids set forth in SEQ ID NO:3 orin corresponding residues in a FVII polypeptide. Exemplary modificationsinclude P54S, Q66N, L121S, A122N, P129A and E132S. In some examples,modified FVII polypeptides containing a modification at a positioncorresponding P54, Q66, L121, A122, P129 or E132 also contain one ormore further modifications, including amino acid replacements,insertions or deletions at another position in the FVII polypeptide.Such modifications include P54S, S52N, Y58S, S119N, G124S, T128N, T130N,V158D, A175S, S222A, G241S, E296V, M298Q, E394N, P395A, R396S, G318N andQ366V. Thus, exemplary of the combination modifications in a FVIIpolypeptide provided herein are S119N/L121S, T128N/P129A, A122N/G124S,A122N/G124S/A175S, A122N/G124S/E394N/P395A/R396S,A122N/G124S/E394N/P395A/R396S/G318N, A122N/G124S/E394N/P395A/R396S,S52N/P54S/A122N/G124S/E394N/P395A/R396S, S52N/P54S, S119N/L121S/A175S,T128N/P129A/A175S, T130N/E132S, Q66N/Y68S,T128N/P129A/V158D/E296V/M298Q, T128N/P129A/S222A,T128N/P129A/A175S/Q366V, A122N/G124S/A175S/Q366V,T128N/P129A/A175S/S222A, A122N/G124S/A175S/S222A, T128N/P129A/M298Q andT128N/P129A/M298Q/H373F.

Also provided herein are modified FVII polypeptides containing amodification corresponding to T239S, T239Q, T239V, T239L, T239H, T239I,P321K, P321E, P321Y, P321S, Q366D, Q366N, Q366V, Q366I, Q366L, Q366M,H373D, H373E, H373S, H373F, H373A, K161S, K161V, H216S, H216K, H216R,S222A, S222K, S222V, S222D, S222N, S222E or H257S in a FVII polypeptidehaving a sequence of amino acids set forth in SEQ ID NO:3 or incorresponding residues in a FVII polypeptide. Further, such modifiedFVII polypeptides also can contain one or more further modifications atanother position, such as amino acid position A51, S52, P54, S60, Q66,Y68, K109, S119, A122, G124, T130, E132, V158, K161, A175, D196, K197,K199, R202, H216, S222, G237, T239, H257, Q286, L287, R290, A292, A294,E296, M298, L305, S314, G318, P321, K337, K341, Q366, H373, F374, E394,P395 and R396. Exemplary modifications at these positions include Q286N,Q286E, Q286D, Q286S, Q286T, Q286R, Q286K, Q286A, Q286V, Q286M, Q286L,Q286Y, D196K, D196R, D196A, D196Y, D196F, D196W, D196L, D196I, K197Y,K197A, K197E, K197D, K197L, K197M, K197I, K197V, K197F, K197W, K199A,K199D, K199E, G237W, G237T, G237I, G237V, T239A, R290A, R290E, R290D,R290N, R290Q, R290K, R290M, R290V, K341E, K341R, K341Q, K341N, K341M,K341D, G237T238insA, G237T238insS, G237T238insV, G237T238insAS,G237T238insSA, D196K197insK, D196K197insR, D196K197insY, D196K197insW,D196K197insA, D196K197insM, K197I198insE, K197I198insY, K197I198insA,K197I198insS, T239S, T239N, T239Q, T239V, T239L, T239H, T239I, L287T,P321K, P321E, P321Y, P321S, Q366D, Q366E, Q366N, Q366T, Q366S, Q366V,Q366I, Q366L, Q366M, H373D, H373E, H373S, H373F, H373A, K161S, K161A,K161V, H216S, H216A, H216K, H216R, S222A, S222K, S222V, S222N, S222E,S222D, H257A, H257S, Gla swap FIX, Gla swap FX, Gla Swap Prot C, GlaSwap Prot S, Gla swap Thrombin, Gla Swap FIX, {Gla Swap FIX/E40L}, {GlaSwap FIX/K43I}, {Gla Swap FIX/Q44S}, {Gla Swap FIX/M19K}, {Gla SwapFIX/M19K/E40L/K43I/Q44S}, S52A, S60A, E394N, P395A, R396S, R202S, A292N,A294S, G318N, A175S, K109N, A122N, G124S, A51N, T130N, E132S, S52N,P54S, S119N, L121S, T128N, P129A, Q66N, Y68S,S103S111delinsQRLMEDICLPRWGCLWEDDF, H115S126delinsQRLMEDICLPRWGCLWEDDF,T128P134delinsQRLMEDICLPRWGCLWEDDF, S103 S111delinsIEDICLPRWGCLWE,H115S126delinsIEDICLPRWGCLWE, T128P134delinsIEDICLPRWGCLWE,S103S111delinsDICLPRWGCLWED, H115S126delinsDICLPRWGCLWED,T128P134delinsDICLPRWGCLWED, P406insIEDICLPRWGCLW,P406insGGGSIEDICLPRWGCLW, P406insDICLPRWGCLWED,P406insGGGSDICLPRWGCLWED, S103S111delinsSFGRGDIRNV,H115S126delinsSFGRGDIRNV, T127P134delinsSFGRGDIRNV, P406insCSFGRGDIRNVC,P406insGGGSCSFGRGDIRNVC, V158T, V158D, L287T, M298K and M298Q. Theresulting combination modifications can include Q366D/H373E,Q366V/H373V, Q366V/H373L, Q366V/H373I, S222K/H257A, H216A/S222A,S222S/Gla Swap FIX, S222A/H257A/Gla Swap FIX, S222A/M298Q,S222A/H257A/M298Q, S222A/A292N/A294S/Q366V, A 175S/S222A/Q366V,S222A/Q366V, H257S/Q366V, S222A/H373A, M298Q/H373F, S52A/S60A/S222A,S222A/T239V, V158D/T239V/E296V/M298Q, S222A/T239I,V158D/E296V/M298Q/H373F, Gla Swap FIX/Q366V, M298Q/Q366N/H373F,T239V/M298Q/H373F and T239I/M298Q/H373F.

Provided herein are modified FVII polypeptides containing two or moremodifications in a FVII polypeptide, allelic and species variant thereofor active fragments thereof, wherein the two or more amino acidmodifications are selected from among amino acid modificationscorresponding to H216A, H257A, E394N, P395A, R396S, K109N, A292N, A175S,H257A and Gla Swap FIX. For example, modified FVII polypeptides providedherein include those with modifications selected from among H216A/H257A,E394N/P395A/R396S and K109N/A175S. Further, a modification correspondingto M298Q or A294S also can be included. Thus, also provided herein aremodified FVII polypeptides containing modifications selected from amongH216A/H257A, E394N/P395A/R396S and K109N/A175S. In some examples, themodified FVII polypeptides also contain a modification corresponding toM298Q or A294S. This can result in, for example, a modified FVIIpolypeptide containing the modifications H257A/M298Q orK109N/A292N/A294S. Also provided are modified FVII polypeptidescontaining modifications corresponding to S52A/S60A/V158D/E296V/M298Q orV158D/T239I/E296V/M298.

In some examples, the modified FVII polypeptides provided herein containa serum albumin binding sequence, such as a sequence of amino acids setforth in any of SEQ ID NOS: 103-109, or a sufficient portion thereof toeffect serum albumin binding. Such modified FVII polypeptides canexhibit increased affinity for or binding to serum albumin bindingcompared with the unmodified FVII polypeptide. For example, modifiedFVII polypeptides containing a serum albumin binding sequence canexhibit at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100%, 200%, 300%, 400%, 500% or more increased affinity for orbinding to serum albumin binding. The serum albumin binding sequence canreplace a contiguous sequence of amino acid residues of the unmodifiedFVII polypeptide. Modified FVII polypeptides containing a serum albuminbinding sequence can contain a modification selected from amongS103S111delinsQRLMEDICLPRWGCLWEDDF, H115S126delinsQRLMEDICLPRWGCLWEDDF,T128P134delinsQRLMEDICLPRWGCLWEDDF, S103S111delinsIEDICLPRWGCLWE,H115S126delinsIEDICLPRWGCLWE, T128P134delinsIEDICLPRWGCLWE,S103S111delinsDICLPRWGCLWED, H115S126delinsDICLPRWGCLWED,T128P134delinsDICLPRWGCLWED, P406insIEDICLPRWGCLW,P406insGGGSIEDICLPRWGCLW, P406insDICLPRWGCLWED andP406insGGGSDICLPRWGCLWED

Provided herein are modified FVII polypeptides containing a plateletintegrin α_(IIb)β₃ binding sequence. Such modified FVII polypeptides canexhibit increased affinity for or binding to platelet integrin α_(IIb)β₃compared with the unmodified FVII polypeptide. For example, modifiedFVII polypeptides containing a platelet integrin α_(IIb)β₃ bindingsequence can exhibit at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more increasedaffinity for or binding to platelet integrin α_(IIb)β₃ binding comparedto the unmodified FVII polypeptide. Examples of serum albumin bindingsequence include those sequence of amino acids set forth in any of SEQID NOS: 110-112, or a sufficient portion thereof to effect plateletintegrin α_(IIb)β₃ binding, which can replace a contiguous sequence ofamino acid residues of the unmodified FVII polypeptide. Modified FVIIpolypeptides containing a modification selected from amongS103S111delinsSFGRGDIRNV, H115S126delinsSFGRGDIRNV,T127P134delinsSFGRGDIRNV, P406insCSFGRGDIRNVC andP406insGGGSCSFGRGDIRNVC.

The modified FVII polypeptides containing a serum albumin or plateletintegrin α_(IIb)β₃ binding sequence also can contain one or more furthermodifications at another position in the FVII polypeptide, such as aminoacid replacement at a position corresponding to a position G237V. Thusalso provided herein are modified FVII polypeptides containing amodification selected from among S103S111delinsIEDICLPRWGCLWE/G237V,S103S111delinsDICLPRWGCLWED/G237V,H115S126delinsQRLMEDICLPRWGCLWEDDF/G237V,H115S126delinsIEDICLPRWGCLWE/G237V, H115S126delinsDICLPRWGCLWED/G237V,T128P34delinsQRLMEDICLPRWGCLWEDDF/G237V,T128P134delinsIEDICLPRWGCLWE/G237V,S103S111delinsQRLMEDICLPRWGCLWEDDF/G237V andT128P134delinsDICLPRWGCLWED/G237V

In some examples, the modified FVII polypeptides provided herein contain2, 3, 4, 5, 6, 7 or more modifications. In further examples, themodified FVII polypeptides provided herein contain a heterologous Gladomain, or a sufficient portion thereof to effect phospholipid binding.Such polypeptides can exhibit increased affinity for or binding tophospholipids compared with the unmodified FVII polypeptide, such as atleast about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,100%, 200%, 300%, 400%, 500% or more increased affinity for or bindingto phospholipids. The heterologous Gla domain can be selected from amonga Gla domain in Factor IX (FIX), Factor X (FX), prothrombin, protein C,protein S, osteocalcin, matrix Gla protein, Growth-arrest-specificprotein 6 (Gas6) and protein Z and, in some examples, can have asequence of amino acids set forth in any of SEQ ID NOS: 83-91, 93 and94, or a sufficient portion thereof to effect phospholipid binding. Allor a contiguous portion of the native FVII Gla domain, which can includeamino acids 1-45 in a FVII polypeptide having a sequence of amino acidsset forth in SEQ ID NO:3, or in corresponding residues in a FVIIpolypeptide, can removed and replaced with the heterologous Gla domain,or a sufficient portion thereof to effect phospholipid binding.

Modified FVII polypeptides provided herein can exhibit increasedresistance to antithrombin III compared with the unmodified FVIIpolypeptide. Such a modified FVII polypeptide can exhibit at least orabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%,300%, 400%, 500% or more resistance to antithrombin III compared to theunmodified FVII polypeptide. The modified FVII polypeptides providedherein also can exhibit increased catalytic or coagulant activitycompared with the unmodified FVII polypeptide, such as an increase ofleast about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%,200%, 300%, 400%, 500% or more compared to an unmodified FVIIpolypeptide. Further, the modified FVII polypeptides provided herein canexhibit increased resistance to TFPI compared with the unmodified FVIIpolypeptide. Such modified FVII polypeptides can be at least or about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%,400%, 500% or more resistant to TFPI compared to an unmodified FVIIpolypeptide. Modified FVII polypeptides provided herein also can exhibitincreased resistance to the inhibitory effects of Zn²⁺ compared with theunmodified FVII polypeptide. For example, a modified FVII polypeptidecan be at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100%, 200%, 300%, 400%, 500% or more resistant to theinhibitory effects of Zn2+ compared to an unmodified FVII polypeptide.

In some examples, the modified FVII polypeptides provided herein containone or more modifications that introduce and/or eliminate one or moreglycosylation sites compared to the unmodified FVII polypeptide. Forexample, 1, 2, 3, 4, 5, 6, or more glycosylation sites can be introducedor eliminated. Glycosylation sites that can be introduced or eliminatedinclude N-glycosylation sites and O-glycosylation sites. The modifiedFVII polypeptides provided herein can contain one or more further aminoacid modification(s) that increases resistance to antithrombin-III,increases binding and/or affinity to phospholipids, increases affinityfor tissue factor, increases intrinsic activity, increases TF-dependentactivity, increases coagulant activity, alters the conformation of thepolypeptide to alter zymogenicity, increases catalytic or coagulantactivity by shifting the equilibrium between highly active and lessactive FVIIa conformations in favor of the highly active conformations,increases resistance to proteases, decreases glycosylation, increasesglycosylation, reduces immunogenicity, increases stability, and/orfacilitates chemical group linkage. In some examples, the alteredzymogenicity confers a more zymogen-like shape or a less zymogen-likeshape.

In some examples, the modified FVII polypeptides provided herein containone or more further amino acid modification(s) selected from amongS279C/V302C, L280C/N301C, V281C/V302C, S282C/V299C, insertion of atyrosine at position 4, F4S, F4T, P10Q, P10E, P10D, P10N, Q21N, R28F,R28E, I30C, I30D, I30E, K32D, K32Q, K32E, K32G, K32H, K32T, K32C, K32A,K32S, D33C, D33F, D33E, D33K, A34C, A34E, A34D, A34I, A34L, A34M, A34V,A34F, A34W, A34Y, R36D, R36E, T37C, T37D, T37E, K38C, K38E, K38T, K38D,K38L, K38G, K38A, K38S, K38N, K38H, L39E, L39Q, L39H, W41N, W41C, W41E,W41D, I42R, I42N, I42S, I42A, I42Q, I42N, I42S, I42A, I42Q, I42K, S43Q,S43N, Y44K, Y44C, Y44D, Y44E, S45C, S45D, S45E, D46C, A51N, S53N, G58N,G59S, G59T, K62E, K62R, K62D, K62N, K62Q, K62T, L65Q, L65S, L65N, F71D,F71Y, F71E, F71Q, F71N, P74S, P74A, A75E, A75D, E77A, E82Q, E82N, E82S,E82T T83K, N95S, N95T, G97S, G97T, Y101N, D104N, T106N, K109N, E116D,G117N, G124N, S126N, T128N, L141C, L141D, L141E, E142D, E142C, K143C,K143D, K143E, R144E, R144C, R144D, N145Y, N145G, N145F, N145M, N145S,N145I, N145L, N145T, N145V, N145P, N145K, N145H, N145Q, N145E, N145R,N145W, N145D, N145C, K157V, K157L, K157I, K157M, K157F, K157W, K157P,K157G, K157S, K157T, K157C, K157Y, K157N, K157E, K157R, K157H, K157D,K157Q, V158L, V158I, V158M, V158F, V158W, V158P, V158G, V158S, V158T,V158C, V158Y, V158N, V158E, V158R, V158K, V158H, V158D, V158Q, A175S,A175T, G179N, I186S, I186T, V188N, R202S, R202T, I205S, I205T, D212N,E220N, I230N, P231N, P236N, G237N, Q250C, V253N, E265N, T267N, E270N,A274M, A274L, A274K, A274R, A274D, A274V, A2741, A274F, A274W, A274P,A274G, A274T, A274C, A274Y, A274N, A274E, A274H, A274S, A274Q, F275H,R277N, F278S, F278A, F278N, F278Q, F278G, L280N, L288K, L288C, L288D,D289C, D289K, L288E, R290C, R290G, R290A, R290S, R290T, R290K, R290D,R290E, G291E, G291D, G291C, G291N, G291K, A292C, A292K, A292D, A292E,T293K, E296V, E296L, E296I, E296M, E296F, E296W, E296P, E296G, E296S,E296T, E296C, E296Y, E296N, E296K, E296R, E296H, E296D, E296Q, M298Q,M298V, M298L, M298I, M298F, M298W, M298P, M298G, M298S, M298T, M298C,M298Y, M298N, M298K, M298R, M298H, M298E, M298D, P303S, P303T, R304Y,R304F, R304L, R304M, R304G, R304T, R304A, R304S, R304N, L305V, L305Y,L305I, L305F, L305A, L305M, L305W, L305P, L305G, L305S, L305T, L305C,L305N, L305E, L305K, L305R, L305H, L305D, L305Q, M306D, M306N, D309S,D309T, Q312N, Q313K, Q313D, Q313E, S314A, S314V, S314I, S314M, S314F,S314W, S314P, S314G, S314L, S314T, S314C, S314Y, S314N, S314E, S314K,S314R, S314H, S314D, S314Q, R315K, R315G, R315A, R315S, R315T, R315Q,R315C, R315D, R315E, K316D, K316C, K316E, V317C, V317K, V317D, V317E,G318N, N322Y, N322G, N322F, N322M, N322S, N322I, N322L, N322T, N322V,N322P, N322K, N322H, N322Q, N322E, N322R, N322W, N322C, G331N, Y332S,Y332A, Y332N, Y332Q, Y332G, D334G, D334E, D334A, D334V, D334I, D334M,D334F, D334W, D334P, D334L, D334T, D334C, D334Y, D334N, D334K, D334R,D334H, D334S, D334Q, S336G, S336E, S336A, S336V, S336I, S336M, S336F,S336W, S336P, S336L, S336T, S336C, S336Y, S336N, S336K, S336R, S336H,S336D, S336Q, K337L, K337V, K337I, K337M, K337F, K337W, K337P, K337G,K337S, K337T, K337C, K337Y, K337N, K337E, K337R, K337H, K337D, K337Q,K341E, K341Q, K341G, K341T, K341A, K341S, G342N, H348N, R353N, Y357N,I361N, F374P, F374A, F374V, F374I, F374L, F374M, F374W, F374G, F374S,F374T, F374C, F374Y, F374N, F374E, F374K, F374R, F374H, F374D, F374Q,V376N, R379N, L390C, L390K, L390D, L390E, M391D, M391C, M391K, M391N,M391E, R392C, R392D, R392E, S393D, S393C, S393K, S393E, E394K, P395K,E394C, P395D, P395C, P395E, R396K, R396C, R396D, R396E, P397D, P397K,P397C, P397E, G398K, G398C, G398D, G398E, V399C, V399D, V399K, V399E,L400K, L401K, L401C, L401D, L401E, R402D, R402C, R402K, R402E, A403K,A403C, A403D, A403E, P404E, P404D, P404C, P404K, F405K, P406C,K32N/A34S, K32N/A34T, F31N/D33S, F31N/D33T, I30N/K32S, I30N/K32T,A34N/R36S, A34N/R36T, K38N/F40S, K38N/F40T, T37N/L39S, T37N/L39T,R36N/K38S, R36N/K38T, L39N/W41S, L39N/W41T, F40N/I42S, F40N/I42T,I42N/Y44S, I42N/Y44T, Y44N/D46S, Y44N/D46T, D46N/D48S, D46N/D48T,G47N/Q49S, G47N/Q49T, K143N/N145S, K143N/N145T, E142N/R144S,E142N/R144T, L141N/K143S, L141N/K143T, I140N/E142S, I140N/E142T,R144N/A146S, R144N/A146T, A146N/K148S, A146N/K148T, S147N/P149S/,S147N/P149T, R290N/A292S, R290N/A292T, D289N/G291S, D289N/G291T,L288N/R290S, L288N/R290T, L287N/D289S, L287N/D289T, A292N/A294S,A292N/A294T, T293N/L295S, T293N/L295T, R315N/V317S, R315N/V317T,S314N/K316S, S314N/K316T, Q313N/R315S, Q313N/R315T, K316N/G318S,K316N/G318T, V317N/D319S, V317N/D319T, K341N/D343S, K341N/D343T,S339N/K341S, S339N/K341T, D343N/G345S, D343N/G345T, R392N/E394S,R392N/E394T, L390N/R392S, L390N/R392T, K389N/M391S, K389N/M391T,S393N/P395S, S393N/P395T, E394N/R396S, E394N/R396T, P395N/P397S,P395N/P397T, R396N/G398S, R396N/G398T, P397N/V399S, P397N/V399T,G398N/L400S, G398N/L400T, V399N/L401S, V399N/L401T, L400N/R402S,L400N/R402T, L401N/A403S, L401N/A403T, R402N/P404S, R402N/P404T,A403N/F405S, A403N/F405T, P404N/P406S and P404N/P406T. In some examples,the modified FVII polypeptides also contain a substitution of positions300-322, 305-322, 300-312, or 305-312 with the corresponding amino acidsfrom trypsin, thrombin or FX, or substitution of positions 310-329,311-322 or 233-329 with the corresponding amino acids from trypsin.

Exemplary of modified FVII polypeptides provided herein are those havinga sequence of amino acids set forth in any of SEQ ID NOS: 113-273. Insome examples, the modifications are made in an unmodified FVIIpolypeptide that is an allelic or species variant of the polypeptide setforth in SEQ ID NO:3. The allelic or species or other variant can have40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to the polypeptide set forth in SEQ ID NO:3,excluding the amino acid modification(s). The modified FVII polypeptideprovided herein can be a human polypeptide, a non-human polypeptideand/or a mature polypeptide. In some examples, only the primary sequenceis modified. In other examples, a chemical modification or apost-translational modification also is included. For example, themodified FVII polypeptide can be glycosylated, carboxylated,hydroxylated, sulfated, phosphorylated, albuminated, or conjugated to apolyethylene glycol (PEG) moiety.

The modified FVII polypeptides provided herein can be single-chainpolypeptides, a two-chain polypeptides and/or active or activated.Activation can be effected by proteolytic cleavage by autoactivation,cleavage by Factor IX (FIXa), cleavage by Factor X (FXa), cleavage byFactor XII (FXIIa), or cleavage by thrombin.

The modified FVII polypeptides provided herein can retain one or moreactivities of the unmodified FVII polypeptide. For example, the modifiedFVII polypeptides can contain modifications at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50 or 60 amino acid positions so long as thepolypeptide retains at least one FVII activity of the unmodified FVIIpolypeptide. Such modified FVII polypeptides can retain at least about1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more of an activityof the unmodified FVII polypeptide. In some examples, one or moreactivities are selected from among tissue factor (TF) binding, factor X(FX) activation, Factor IX (FIX) activation, phospholipid binding, andcoagulation activity. Further, the activities that are retained canincreased or decreased compared to the unmodified FVII polypeptide. Insome examples, the coagulation activity is increased compared to theunmodified FVII polypeptide, such as at least about 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or moreof the coagulation activity of the unmodified FVII polypeptide.Activities can measured in vitro, ex vivo or in vivo.

Provided herein are nucleic acid molecules containing a sequence ofnucleotides encoding modified FVII polypeptides provided herein. Alsoprovided are vectors containing such nucleic acid molecules, includingprokaryotic vectors, viral vectors, or a eukaryotic vectors, such as amammalian vector. Viral vectors can selected from among an adenovirus,an adeno-associated-virus, a retrovirus, a herpes virus, a lentivirus, apoxvirus, and a cytomegalovirus. Provided herein are cells containingthese vectors, including eukaryotic cells, such as mammalian cells.Exemplary of mammalian cells are baby hamster kidney cells (BHK-21) or293 cells or CHO cells. In some examples, the cells express the modifiedFVII polypeptide. Thus, also provided herein are modified FVIIpolypeptides that are produced by these cells.

Provided herein are pharmaceutical compositions containing atherapeutically effective concentration or amount of any modified FVIIpolypeptide, nucleic acid molecule, vector or cell provided herein in apharmaceutically acceptable vehicle. In some examples, thepharmaceutical composition is formulated for local, systemic, or topicaladministration, such as oral, nasal, pulmonary buccal, transdermal,subcutaneous, intraduodenal, enteral, parenteral, intravenous, orintramuscular administration. The pharmaceutical compositions also canbe formulated for controlled-release and/or single-dosageadministration.

Provided herein are methods of treating a subject by administering apharmaceutical composition provided herein, wherein the subject has adisease or condition that is treated by administration of FVII or aprocoagulant, such as by administration of active FVII (FVIIa). In someexamples, treatment with the pharmaceutical composition ameliorates oralleviates the symptoms associated with the disease or condition. Infurther examples, the methods provided herein also include monitoringthe subject for changes in the symptoms associated with disease orcondition that is treated by administration of FVII or a procoagulant.The disease or condition to be treated using the methods provided hereincan be selected from among blood coagulation disorders, hematologicdisorders, hemorrhagic disorders, hemophilia (such as is hemophilia A orhemophilia B or hemophilia C, congenital or acquired hemophilia), factorVII deficiency and bleeding disorders, including bleeding complicationdue to surgery (such as heart surgery, angioplasty, lung surgery,abdominal surgery, spinal surgery, brain surgery, vascular surgery,dental surgery, or organ transplant surgery) or trauma. In someexamples, the bleeding is manifested as acute haemarthroses, chronichaemophilic arthropathy, haematomas, haematuria, central nervous systembleedings, gastrointestinal bleedings, or cerebral haemorrhage. Infurther examples, the bleeding is due to dental extraction. Thetransplant surgery can be selected from among transplantation of bonemarrow, heart, lung, pancreas, and liver.

In some examples, the method provided herein can be used to treat asubject that has autoantibodies to factor VIII or factor IX. The methodsprovided herein also can included administering one or more additionalcoagulation factors, such as plasma purified or recombinant coagulationfactors, procoagulants, such as vitamin K, vitamin K derivative andprotein C inhibitors, plasma, platelets, red blood cells andcorticosteroids, or treatments.

Provided herein are articles of manufacture containing packagingmaterial and a pharmaceutical composition provided herein containedwithin the packaging material. In some examples, the modified FVIIpolypeptide in the pharmaceutical composition is effective for treatmentof a FVII-mediated disease or disorder, and the packaging materialincludes a label that indicates that the modified FVII polypeptide isused for treatment of a FVII-mediated disease or disorder. Also providedherein are kits, comprising a pharmaceutical composition describedherein, a device for administration of the composition and, optionally,instructions for administration.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the coagulation cascade. The figure shows the intrinsicpathway and the extrinsic pathway of coagulation for the independentproduction of FXa and convergence of the pathways to a common pathway togenerate thrombin and fibrin for the formation of a clot. These pathwaysare interconnected. The figure depicts the order of molecules involvedin the activation cascade in which a zymogen is converted to anactivated protease by cleavage of one or more peptide bonds. Theactivated protease then serves as the activating protease for the nextzymogen molecule in the cascade, ultimately resulting in clot formation.

FIG. 2 depicts the cell based model of coagulation (see e.g. Hoffman etal. (2001) Thromb Haemost 85:958-965). The figure depicts thecoagulation events as being separated into three phases, whereinitiation of coagulation is effected by the activation of FX to FXa bythe TF/FVIIa complex on the TF-bearing cell, resulting in the generationof a small amount of thrombin after activation by FXa/FVa. Amplificationtakes place when thrombin binds to and activates the platelets, andinitiates the activation of sufficient quantities of the appropriatecoagulation factors to form the FVIIIa/FIXa and FVa/FXa complexes.Propagation of coagulation occurs on the surface of large numbers ofactivated platelets at the site of injury, resulting in a burst ofthrombin generation that is sufficiently large to generate enough fibrinfrom fibrinogen to establish a clot at the site of injury.

FIG. 3 depicts the mechanisms by which FVIIa can initiate thrombinformation. The figure illustrates the TF-dependent pathway of FVIIathrombin generation, which acts at the surface of a TF-bearing cell andinvolves complexing of FVIIa with TF prior to activation of FX to FXa.The figure also depicts the TF-independent pathway of FVIIa thrombingeneration, during which FVIIa binds to phospholipids on the activatedplatelet and activates FX to FXa, which in turn complexes with FVa tocleave prothrombin into thrombin.

DETAILED DESCRIPTION

Outline A. Definitions B. Hemostasis Overview 1. Platelet adhesion andaggregation 2. Coagulation cascade a. Initiation b. Amplification c.Propagation 3. Regulation of Coagulation C. Factor VII (FVII) 1. FVIIstructure and organization 2. Post-translational modifications 3. FVIIprocessing 4. FVII activation 5. FVII function a. Tissuefactor-dependent FVIIa activity b. Tissue factor-independent FVIIaactivity 6. FVII as a biopharmaceutical D. Modified FVII polypeptides 1.Increased catalytic activity a. Exemplary modifications to increasecatalytic activity i. Basic amino acid substitutions at position 286 ii.Other mutations at position 286 2. Increased resistance to AT-IIIExemplary modifications to effect increased resistance to AT-III 3.Increased resistance to inhibition by Zn²⁺ Exemplary modifications toincrease resistance to inhibition by Zn²⁺ 4. Altered glycosylationExemplary modifications to alter glycosylation 5. Increased binding toserum albumin and/or platelet integrin α_(IIb)β₃ a. Exemplary FVIIpolypeptides with serum albumin binding sequences b. Exemplary FVIIpolypeptides with platelet integrin α_(IIb)β₃ binding sequences 6.Modification by introduction of a heterologous Gla domain 7.Combinations and Additional Modifications a. Modifications that increaseresistance to TFPI b. Modifications that increase intrinsic activity c.Modifications that increase resistance to proteases d. Modificationsthat increase affinity for phospholipids e. Modifications that alterglycosylation f. Modifications to facilitate chemical group linkage g.Exemplary combination mutations E. Production of FVII polypeptides 1.Vectors and cells 2. Expression systems a. Prokaryotic expression b.Yeast c. Insects and insect cells d. Mammalian cells e. Plants 2.Purification 3. Fusion proteins 4. Polypeptide modifications 5.Nucleotide sequences F. Assessing modified FVII polypeptideactivities 1. In vitro assays a. Post-translational modification b.Proteolytic activity c. Coagulation activity d. Binding to and/orinhibition by other proteins e. Phospholipid binding 2. Non-human animalmodels 3. Clinical assays G. Formulation and administration 1.Formulations a. Dosages b. Dosage forms 2. Administration of modifiedFVII polypeptides 3. Administration of nucleic acids encoding modifiedFVII polypeptides (gene therapy) H. Therapeutic Uses 1. Congenitalbleeding disorders a. Hemophilia b. FVII deficiency c. Others 2.Acquired bleeding disorders a. Chemotherapy-acquired thrombocytopenia b.Other coagulopathies c. Transplant-acquired bleeding d. Anticoagulanttherapy-induced bleeding e. Acquired hemophilia 3. Trauma and surgicalbleeding I. Combination Therapies J. Articles of manufacture and kits K.Examples

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, Genbank sequences, databases,websites and other published materials referred to throughout the entiredisclosure herein, unless noted otherwise, are incorporated by referencein their entirety. In the event that there are a plurality ofdefinitions for terms herein, those in this section prevail. Wherereference is made to a URL or other such identifier or address, itunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, coagulation pathway or coagulation cascade refers to theseries of activation events that leads to the formation of an insolublefibrin clot. In the coagulation cascade or pathway, an inactive proteinof a serine protease (also called a zymogen) is converted to an activeprotease by cleavage of one or more peptide bonds, which then serves asthe activating protease for the next zymogen molecule in the cascade. Inthe final proteolytic step of the cascade, fibrinogen is proteolyticallycleaved by thrombin to fibrin, which is then crosslinked at the site ofinjury to form a clot.

As used herein, “hemostasis” refers to the stopping of bleeding or bloodflow in an organ or body part. The term hemostasis can encompass theentire process of blood clotting to prevent blood loss following bloodvessel injury to subsequent dissolution of the blood clot followingtissue repair.

As used herein, “clotting” or “coagulation” refers to the formation ofan insoluble fibrin clot, or the process by which the coagulationfactors of the blood interact in the coagulation cascade, ultimatelyresulting in the formation of an insoluble fibrin clot.

As used herein, a “protease” is an enzyme that catalyzes the hydrolysisof covalent peptidic bonds. These designations include zymogen forms andactivated single-, two- and multiple-chain forms thereof. For clarity,reference to proteases refer to all forms. Proteases include, forexample, serine proteases, cysteine proteases, aspartic proteases,threonine and metallo-proteases depending on the catalytic activity oftheir active site and mechanism of cleaving peptide bonds of a targetsubstrate.

As used herein, serine proteases or serine endopeptidases refers to aclass of peptidases, which are characterized by the presence of a serineresidue in the active site of the enzyme. Serine proteases participatein a wide range of functions in the body, including blood clotting andinflammation, as well as functioning as digestive enzymes in prokaryotesand eukaryotes. The mechanism of cleavage by serine proteases is basedon nucleophilic attack of a targeted peptidic bond by a serine.Cysteine, threonine or water molecules associated with aspartate ormetals also can play this role. Aligned side chains of serine, histidineand aspartate form a catalytic triad common to most serine proteases.The active site of serine proteases is shaped as a cleft where thepolypeptide substrate binds.

As used herein, Factor VII (FVII, F7; also referred to as Factor 7,coagulation factor VII, serum factor VII, serum prothrombin conversionaccelerator, SPCA, proconvertin and eptacog alpha) refers to a serineprotease that is part of the coagulation cascade. FVII includes a Gladomain, two EGF domains (EGF-1 and EGF-2), and a serine protease domain(or peptidase S1 domain) that is highly conserved among all members ofthe peptidase S1 family of serine proteases, such as for example withchymotrypsin. The sequence of an exemplary precursor FVII having asignal peptide and propeptide is set forth in SEQ ID NO:1. An exemplarymature FVII polypeptide is set forth in SEQ ID NO:3. FVII occurs as asingle chain zymogen, a zymogen-like two-chain polypeptide and a fullyactivated two-chain form. Full activation, which occurs uponconformational change from a zymogen-like form, occurs upon binding tois co-factor tissue factor. Also, mutations can be introduced thatresult in the conformation change in the absence of tissue factor.Hence, reference to FVII includes single-chain and two-chain formsthereof, including zymogen-like and fully activated two-chain forms.

Reference to FVII polypeptide also includes precursor polypeptides andmature FVII polypeptides in single-chain or two-chain forms, truncatedforms thereof that have activity, and includes allelic variants andspecies variants, variants encoded by splice variants, and othervariants, including polypeptides that have at least 40%, 45%, 50%, 55%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to the precursor polypeptide set forth in SEQ ID NO:1 or themature form thereof. Included are modified FVII polypeptides, such asthose of SEQ ID NOS: 113 and 273 and variants thereof. Also included arethose that retain at least an activity of a FVII, such as TF binding,factor X binding, phospholipid binding, and/or coagulant activity of aFVII. By retaining activity, the activity can be altered, such asreduced or increased, as compared to a wild-type FVII so long as thelevel of activity retained is sufficient to yield a detectable effect.FVII polypeptides include, but are not limited to, tissue-specificisoforms and allelic variants thereof, synthetic molecules prepared bytranslation of nucleic acids, proteins generated by chemical synthesis,such as syntheses that include ligation of shorter polypeptides, throughrecombinant methods, proteins isolated from human and non-human tissueand cells, chimeric FVII polypeptides and modified forms thereof. FVIIpolypeptides also include fragments or portions of FVII that are ofsufficient length or include appropriate regions to retain at least oneactivity (upon activation if needed) of a full-length maturepolypeptide. FVII polypeptides also include those that contain chemicalor posttranslational modifications and those that do not containchemical or posttranslational modifications. Such modifications include,but are not limited to, pegylation, albumination, glycosylation,farnysylation, carboxylation, hydroxylation, phosphorylation, and otherpolypeptide modifications known in the art.

Exemplary FVII polypeptides are those of mammalian, including human,origin. Exemplary amino acid sequences of FVII of human origin are setforth in SEQ ID NOS: 1, 2, and 3. Exemplary variants of such a humanFVII polypeptide, include any of the precursor polypeptides set forth inSEQ ID NOS: 18-74. FVII polypeptides also include any of non-humanorigin including, but not limited to, murine, canine, feline, leporine,avian, bovine, ovine, porcine, equine, piscine, ranine, and otherprimate factor VII polypeptides. Exemplary FVII polypeptides ofnon-human origin include, for example, cow (Bos taurus, SEQ ID NO:4),mouse (Mus musculus, SEQ ID NO:5), pygmy chimpanzee (Pan paniscus, SEQID NO:6), chimpanzee (Pan troglodytes, SEQ ID NO:7), rabbit (Oryctolaguscuniculus, SEQ ID NO:8), rat (Rattus norvegicus, SEQ ID NO:9), rhesusmacaque (Macaca mulatta, SEQ ID NO:10), pig (Sus scrofa, SEQ ID NO:11),dog (Canis familiaris, SEQ ID NO:12), zebrafish (Brachydanio rerio, SEQID NO:13), Japanese pufferfish (Fugu rubripes, SEQ ID NO:14), chicken(Gallus gallus, SEQ ID NO:15), orangutan (Pongo pygmaeus, SEQ ID NO:16)and gorilla (Gorilla gorilla, SEQ ID NO:17).

One of skill in the art recognizes that the referenced positions of themature factor VII polypeptide (SEQ ID NO:3) differ by 60 amino acidresidues when compared to the isoform a precursor FVII polypeptide setforth in SEQ ID NO:1, which is the isoform a factor VII polypeptidecontaining the signal peptide and propeptide sequences. Thus, the firstamino acid residue of SEQ ID NO:3 “corresponds to” the sixty first(61st) amino acid residue of SEQ ID NO:1. One of skill in the art alsorecognizes that the referenced positions of the mature factor VIIpolypeptide (SEQ ID NO:3) differ by 38 amino acid residues when comparedto the precursor FVII polypeptide set forth in SEQ ID NO:2, which is theisoform b factor VII polypeptide containing the signal peptide andpropeptide sequences. Thus, the first amino acid residue of SEQ ID NO:3“corresponds to” the thirty-ninth (39^(th)) amino acid residue of SEQ IDNO:2.

As used herein, corresponding residues refers to residues that occur ataligned loci. Related or variant polypeptides are aligned by any methodknown to those of skill in the art. Such methods typically maximizematches, and include methods such as using manual alignments and byusing the numerous alignment programs available (for example, BLASTP)and others known to those of skill in the art. By aligning the sequencesof polypeptides, one skilled in the art can identify correspondingresidues, using conserved and identical amino acid residues as guides.For example, by aligning the sequences of factor VII polypeptides, oneof skill in the art can identify corresponding residues, using conservedand identical amino acid residues as guides. For example, the alanine inamino acid position 1 (A1) of SEQ ID NO:3 (mature factor VII)corresponds to the alanine in amino acid position 61 (A61) of SEQ IDNO:1, and the alanine in amino acid position 39 (A39) of SEQ ID NO:2. Inother instances, corresponding regions can be identified. For example,the Gla domain corresponds to amino acid positions A1 through F45 of SEQID NO:3, to amino acid positions A61 through S105 of SEQ ID NO:1 and toamino acid positions A39 to S83 of SEQ ID NO:2. One skilled in the artalso can employ conserved amino acid residues as guides to findcorresponding amino acid residues between and among human and non-humansequences. For example, amino acid residues S43 and E163 of SEQ ID NO:3(human) correspond to S83 and E203 of SEQ ID NO:4 (bovine).Corresponding positions also can be based on structural alignments, forexample by using computer simulated alignments of protein structure. Inother instances, corresponding regions can be identified.

As used herein, a “proregion,” “propeptide,” or “pro sequence,” refersto a region or a segment that is cleaved to produce a mature protein.This can include segments that function to suppress proteolytic activityby masking the catalytic machinery and thus preventing formation of thecatalytic intermediate (i.e., by sterically occluding the substratebinding site). A proregion is a sequence of amino acids positioned atthe amino terminus of a mature biologically active polypeptide and canbe as little as a few amino acids or can be a multidomain structure.

As used herein, “mature factor VII” refers to a FVII polypeptide thatlacks a signal sequence and a propeptide sequence. Typically, a signalsequence targets a protein for secretion via the endoplasmic reticulum(ER)-golgi pathway and is cleaved following insertion into the ER duringtranslation. A propeptide sequence typically functions inpost-translational modification of the protein and is cleaved prior tosecretion of the protein from the cell. Thus, a mature FVII polypeptideis typically a secreted protein. In one example, a mature human FVIIpolypeptide is set forth in SEQ ID NO:3. The amino acid sequence setforth in SEQ ID NO:3 differs from that of the precursor polypeptides setforth in SEQ ID NOS:1 and 2 in that SEQ ID NO:3 is lacking the signalsequence, which corresponds to amino acid residues 1-20 of SEQ ID NOS:1and 2; and also lacks the propeptide sequence, which corresponds toamino acid residues 21-60 of SEQ ID NO:1 and amino acid residues 21-38of SEQ ID NO:2. Reference to a mature FVII polypeptide encompasses thesingle-chain zymogen form and the two-chain form.

As used herein, “wild-type” or “native” with reference to FVII refers toa FVII polypeptide encoded by a native or naturally occurring FVII gene,including allelic variants, that is present in an organism, including ahuman and other animals, in nature. Reference to wild-type factor VIIwithout reference to a species is intended to encompass any species of awild-type factor VII. Included among wild-type FVII polypeptides are theencoded precursor polypeptide, fragments thereof, and processed formsthereof, such as a mature form lacking the signal peptide as well as anypre- or post-translationally processed or modified forms thereof. Alsoincluded among native FVII polypeptides are those that arepost-translationally modified, including, but not limited to,modification by glycosylation, carboxylation and hydroxylation. NativeFVII polypeptides also include single-chain and two-chain forms. Forexample, humans express native FVII. The amino acid sequence ofexemplary wild-type human FVII are set forth in SEQ ID NOS:1, 2, 3 andallelic variants set forth in SEQ ID NOS:44-100 and the mature formsthereof. Other animals produce native FVII, including, but not limitedto, cow (Bos Taurus, SEQ ID NO:4), mouse (Mus musculus, SEQ ID NO:5),pygmy chimpanzee (Pan paniscus, SEQ ID NO:6), chimpanzee (Pantroglodytes, SEQ ID NO:7), rabbit (Oryctolagus cuniculus, SEQ ID NO:8),rat (Rattus norvegicus, SEQ ID NO:9), rhesus macaque (Macaca mulatta,SEQ ID NO:10), pig (Sus scrofa, SEQ ID NO:11), dog (Canis familiaris,SEQ ID NO:12), zebrafish (Brachydanio rerio, SEQ ID NO:13) Japanesepufferfish (Fugu rubripes, SEQ ID NO:14), chicken (Gallus gallus, SEQ IDNO:15), orangutan (Pongo pygmaeus, SEQ ID NO:16) and gorilla (Gorillagorilla, SEQ ID NO:17).

As used herein, species variants refer to variants in polypeptides amongdifferent species, including different mammalian species, such as mouseand human.

As used herein, allelic variants refer to variations in proteins amongmembers of the same species.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA.

As used herein, a zymogen refers to a protease that is activated byproteolytic cleavage, including maturation cleavage, such as activationcleavage, and/or complex formation with other protein(s) and/orcofactor(s). A zymogen is an inactive precursor of a proteolytic enzyme.Such precursors are generally larger, although not necessarily larger,than the active form. With reference to serine proteases, zymogens areconverted to active enzymes by specific cleavage, including catalyticand autocatalytic cleavage, or by binding of an activating co-factor,which generates an active enzyme. For example, generally, zymogens arepresent in a single-chain form. Zymogens, generally, are inactive andcan be converted to mature active polypeptides by catalytic orautocatalytic cleavage at one or more proteolytic sites to generate amulti-chain, such as a two-chain, polypeptide. A zymogen, thus, is anenzymatically inactive protein that is converted to a proteolytic enzymeby the action of an activator. Cleavage can be effected byautoactivation. A number of coagulation proteins are zymogens; they areinactive, but become cleaved and activated upon the initiation of thecoagulation system following vascular damage. With reference to FVII,the FVII polypeptides exist in the blood plasma as zymogens untilcleavage by aproteases, such as for example, activated factor IX (FIXa),activated factor X (FXa), activated factor XII (FXIIa), thrombin, or byautoactivation to produce a zymogen-like two-chain form, which thenrequires further conformation change for full activity.

As used herein, a “zymogen-like” protein or polypeptide refers to aprotein that has been activated by proteolytic cleavage, but stillexhibits properties that are associated with a zymogen, such as, forexample, low or no activity, or a conformation that resembles theconformation of the zymogen form of the protein. For example, when it isnot bound to tissue factor, the two-chain activated form of FVII is azymogen-like protein; it retains a conformation similar to the uncleavedFVII zymogen, and, thus, exhibits very low activity. Upon binding totissue factor, the two-chain activated form of FVII undergoesconformational change and acquires its full activity as a coagulationfactor.

As used herein, an activation sequence refers to a sequence of aminoacids in a zymogen that is the site required for activation cleavage ormaturation cleavage to form an active protease. Cleavage of anactivation sequence can be catalyzed autocatalytically or by activatingpartners.

As used herein, activation cleavage is a type of maturation cleavage,which induces a conformation change that is required for the developmentof full enzymatic activity. This is a classical activation pathway, forexample, for serine proteases in which a cleavage generates a newN-terminus that interacts with the conserved regions of the protease,such as Asp 194 in chymotrypsin, to induce conformational changesrequired for activity. Activation can result in production ofmulti-chain forms of the proteases. In some instances, single chainforms of the protease can exhibit proteolytic activity.

As used herein, “activated Factor VII” or “FVIIa” refers to anytwo-chain form of a FVII polypeptide. A two-chain form typically resultsfrom proteolytic cleavage, but can be produced synthetically. ActivatedFactor VII, thus, includes the zymogen-like two-chain form with lowcoagulant activity, a fully activated form (about 1000-fold moreactivity) that occurs upon binding to tissue factor, and mutated formsthat exist in a fully activated two-chain form or undergo conformationchange to a fully activated form. For example, a single-chain form ofFVII polypeptide (see, e.g., SEQ ID NO:3) is proteolytically cleavedbetween amino acid residues R152 and I153 of the mature FVIIpolypeptide. The cleavage products, FVII heavy chain and FVII lightchain, which are held together by a disulfide bond (between amino acidresidues C135 and C262 in the FVII of SEQ ID NO:3), form the two-chainactivated FVII enzyme. Proteolytic cleavage can be carried out, forexample, by activated factor IX (FIXa), activated factor X (FXa),activated factor XII (FXIIa), thrombin, or by autoactivation.

As used herein, a “property” of a FVII polypeptide refers to a physicalor structural property, such three-dimensional structure, pI, half-life,conformation and other such physical characteristics.

As used herein, an “activity” of a FVII polypeptide refers to anyactivity exhibited by a factor VII polypeptide. Such activities can betested in vitro and/or in vivo and include, but are not limited to,coagulation or coagulant activity, pro-coagulant activity, proteolyticor catalytic activity such as to effect factor X (FX) activation orFactor IX (FIX) activation; antigenicity (ability to bind to or competewith a polypeptide for binding to an anti-FVII antibody); ability tobind tissue factor, factor X or factor IX; and/or ability to bind tophospholipids. Activity can be assessed in vitro or in vivo usingrecognized assays, for example, by measuring coagulation in vitro or invivo. The results of such assays indicate that a polypeptide exhibits anactivity that can be correlated to activity of the polypeptide in vivo,in which in vivo activity can be referred to as biological activity.Assays to determine functionality or activity of modified forms of FVIIare known to those of skill in the art. Exemplary assays to assess theactivity of a FVII polypeptide include prothromboplastin time (PT) assayor the activated partial thromboplastin time (aPTT) assay to assesscoagulant activity, or chromogenic assays using synthetic substrates,such as described in the Examples, below, to assess catalytic orproteolytic activity.

As used herein, “exhibits at least one activity” or “retains at leastone activity” refers to the activity exhibited by a modified FVIIpolypeptide as compared to an unmodified FVII polypeptide of the sameform and under the same conditions. For example, a modified FVIIpolypeptide in a two-chain form is compared with an unmodified FVIIpolypeptide in a two-chain form, under the same experimental conditions,where the only difference between the two polypeptides is themodification under study. In another example, a modified FVIIpolypeptide in a single-chain form is compared with an unmodified FVIIpolypeptide in a single-chain form, under the same experimentalconditions, where the only difference between the two polypeptides isthe modification under study. Typically, a modified FVII polypeptidethat retains or exhibits at least one activity of an unmodified FVIIpolypeptide of the same form retains a sufficient amount of the activitysuch that, when administered in vivo, the modified FVII polypeptide istherapeutically effective as a procoagulant therapeutic. Generally, fora modified FVII polypeptide to retain therapeutic efficacy as aprocoagulant, the amount of activity that is retained is or is about0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,300%, 400%, 500% or more of the activity of an unmodified FVIIpolypeptide of the same form that displays therapeutic efficacy as aprocoagulant. The amount of activity that is required to maintaintherapeutic efficacy as a procoagulant can be empirically determined, ifnecessary. Typically, retention of 0.5% to 20%, 0.5% to 10%, 0.5% to 5%of an activity is sufficient to retain therapeutic efficacy as aprocoagulant in vivo.

It is understood that the activity being exhibited or retained by amodified FVII polypeptide can be any activity, including, but notlimited to, coagulation or coagulant activity, pro-coagulant activity;proteolytic or catalytic activity such as to effect factor X (FX)activation or Factor IX (FIX) activation; antigenicity (ability to bindto or compete with a polypeptide for binding to an anti-FVII antibody);ability to bind tissue factor, factor X or factor IX; and/or ability tobind to phospholipids. In some instances, a modified FVII polypeptidecan retain an activity that is increased compared to an unmodified FVIIpolypeptide. In some cases, a modified FVII polypeptide can retain anactivity that is decreased compared to an unmodified FVII polypeptide.Activity of a modified FVII polypeptide can be any level of percentageof activity of the unmodified polypeptide, where both polypeptides arein the same form, including but not limited to, 1% of the activity, 2%,3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, 100%, 200%, 300%, 400%, 500%, or more activity compared to thepolypeptide that does not contain the modification at issue. Forexample, a modified FVII polypeptide can exhibit increased or decreasedactivity compared to the unmodified FVII polypeptide in the same form.For example, it can retain at least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%or at least 99% of the activity of the unmodified FVII polypeptide. Inother embodiments, the change in activity is at least about 2 times, 3times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times,20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times,700 times, 800 times, 900 times, 1000 times, or more times greater thanunmodified FVII. The particular level to be retained is a function ofthe intended use of the polypeptide and can be empirically determined.Activity can be measured, for example, using in vitro or in vivo assayssuch as those described herein or in the Examples below.

As used herein, “coagulation activity” or “coagulant activity” or“pro-coagulant activity” refers to the ability of a polypeptide toeffect coagulation. Assays to assess coagulant activity are known tothose of skill in the art, and include prothromboplastin time (PT) assayor the activated partial thromboplastin time (aPTT) assay.

As used herein, “catalytic activity” or “proteolytic activity” withreference to FVII refers to the ability of a FVII protein to catalyzethe proteolytic cleavage of a substrate, and are used interchangeably.Assays to assess such activities are known in the art. For example, theproteolytic activity of FVII can be measured using chromogenicsubstrates such as Spectrozyme FVIIa (CH₃SO₂-D-CHA-But-Arg-pNA), wherecleavage of the substrate is monitored by absorbance and the rate ofsubstrate hydrolysis determined by linear regression.

As used herein, “intrinsic activity” with reference to FVII refers tothe catalytic, proteolytic, and/or coagulant activity of a FVII proteinin the absence of tissue factor.

As used herein, domain (typically a sequence of three or more, generally5 or 7 or more amino acids) refers to a portion of a molecule, such asproteins or the encoding nucleic acids, that is structurally and/orfunctionally distinct from other portions of the molecule and isidentifiable. For example, domains include those portions of apolypeptide chain that can form an independently folded structure withina protein made up of one or more structural motifs and/or that isrecognized by virtue of a functional activity, such as proteolyticactivity. A protein can have one, or more than one, distinct domains.For example, a domain can be identified, defined or distinguished byhomology of the sequence therein to related family members, such ashomology to motifs that define a protease domain or a gla domain. Inanother example, a domain can be distinguished by its function, such asby proteolytic activity, or an ability to interact with a biomolecule,such as DNA binding, ligand binding, and dimerization. A domainindependently can exhibit a biological function or activity such thatthe domain independently or fused to another molecule can perform anactivity, such as, for example proteolytic activity or ligand binding. Adomain can be a linear sequence of amino acids or a non-linear sequenceof amino acids. Many polypeptides contain a plurality of domains. Suchdomains are known, and can be identified by those of skill in the art.For exemplification herein, definitions are provided, but it isunderstood that it is well within the skill in the art to recognizeparticular domains by name. If needed appropriate software can beemployed to identify domains.

As used herein, a protease domain is the catalytically active portion ofa protease. Reference to a protease domain of a protease includes thesingle, two- and multi-chain forms of any of these proteins. A proteasedomain of a protein contains all of the requisite properties of thatprotein required for its proteolytic activity, such as for example, thecatalytic center. In reference to FVII, the protease domain shareshomology and structural feature with the chymotrypsin/trypsin familyprotease domains, including the catalytic triad. For example, in themature FVII polypeptide set forth in SEQ ID NO:3, the protease domaincorresponds to amino acid positions 153 to 392.

As used herein, a gamma-carboxyglutamate (Gla) domain refers to theportion of a protein, for example a vitamin K-dependent protein, thatcontains post-translational modifications of glutamate residues,generally most, but not all of the glutamate residues, by vitaminK-dependent carboxylation to form Gla. The Gla domain is responsible forthe high-affinity binding of calcium ions and binding tonegatively-charged phospholipids. Typically, the Gla domain starts atthe N-terminal extremity of the mature form of vitamin K-dependentproteins and ends with a conserved aromatic residue. In a mature FVIIpolypeptide the Gla domain corresponds to amino acid positions 1 to 45of the exemplary polypeptide set forth in SEQ ID NO:3. Gla domains arewell known and their locus can be identified in particular polypeptides.The Gla domains of the various vitamin K-dependent proteins sharesequence, structural and functional homology, including the clusteringof N-terminal hydrophobic residues into a hydrophobic patch thatmediates interaction with negatively charged phospholipids on the cellsurface membrane. Exemplary other Gla-containing polypeptides include,but are not limited to, FIX, FX, prothrombin, protein C, protein S,osteocalcin, matrix Gla protein, Growth-arrest-specific protein 6(Gas6), and protein Z. The Gla domains of these and other exemplaryproteins are set forth in any of SEQ ID NOS: 83-94.

As used herein, “native” or “endogenous” with reference to a Gla domainrefers to the naturally occurring Gla domain associated with all or apart of a polypeptide having a Gla domain. For purposes herein, a nativeGla domain is with reference to a FVII polypeptide. For example, thenative Gla domain of FVII, set forth in SEQ ID NO:92, corresponds toamino acids 1-45 of the sequence of amino acids set forth in SEQ IDNO:3.

As used herein, a heterologous Gla domain refers to the Gla domain froma polypeptide, from the same or different species, that is not a FVIIGla domain. Exemplary of heterologous Gla domains are the Gla domainsfrom Gla-containing polypeptides including, but are not limited to, FIX,FX, prothrombin, protein C, protein S, osteocalcin, matrix Gla protein,Growth-arrest-specific protein 6 (Gas6), and protein Z. The Gla domainsof these and other exemplary proteins are set forth in any of SEQ IDNOS: 83-91, 93 and 94.

As used herein, a contiguous portion of a Gla domain refers to at leasttwo or more adjacent amino acids, typically 2, 3, 4, 5, 6, 8, 10, 15,20, 30, 40 or more up to all amino acids that make up a Gla domain.

As used herein, “a sufficient portion of a Gla domain to effectphospholipid binding” includes at least one amino acid, typically, 2, 3,4, 5, 6, 8, 10, 15 or more amino acids of the domain, but fewer than allof the amino acids that make up the domain so long as the polypeptidethat contains such portion exhibits phospholipid binding.

As used herein, “replace” with respect to a Gla domain or “Gla domainswap” refers to the process by which the endogenous Gla domain of aprotein is replaced, using recombinant, synthetic or other methods, withthe Gla domain of another protein. In the context of a “Gla domainswap”, a “Gla domain” is any selection of amino acids from a Gla domainand adjacent regions that is sufficient to retain phospholipid bindingactivity. Typically, a Gla domain swap will involve the replacement ofbetween 40 and 50 amino acids of the endogenous protein with between 40and 50 amino acids of another protein, but can involve fewer or moreamino acids.

As used herein, an epidermal growth factor (EGF) domain (EGF-1 or EGF-2)refers to the portion of a protein that shares sequence homology to aspecific 30 to 40 amino acid portion of the epidermal growth factor(EGF) sequence. The EGF domain includes six cysteine residues that havebeen shown (in EGF) to be involved in disulfide bonds. The mainstructure of an EGF domain is a two-stranded beta-sheet followed by aloop to a C-terminal short two-stranded sheet. FVII contains two EGFdomains: EGF-1 and EGF-2. These domains correspond to amino acidpositions 46-82, and 87-128, respectively, of the mature FVIIpolypeptide set forth in SEQ ID NO:3.

As used herein, “unmodified polypeptide” or “unmodified FVII” andgrammatical variations thereof refer to a starting polypeptide that isselected for modification as provided herein. The starting polypeptidecan be a naturally-occurring, wild-type form of a polypeptide. Inaddition, the starting polypeptide can be altered or mutated, such thatit differs from a native wild type isoform but is nonetheless referredto herein as a starting unmodified polypeptide relative to thesubsequently modified polypeptides produced herein. Thus, existingproteins known in the art that have been modified to have a desiredincrease or decrease in a particular activity or property compared to anunmodified reference protein can be selected and used as the startingunmodified polypeptide. For example, a protein that has been modifiedfrom its native form by one or more single amino acid changes andpossesses either an increase or decrease in a desired property, such asa change in a amino acid residue or residues to alter glycosylation, canbe a target protein, referred to herein as unmodified, for furthermodification of either the same or a different property. Exemplarymodified FVII polypeptides known in the art include any FVII polypeptidedescribed in, for example, U.S. Pat. Nos. 5,580,560, 6,017,882,6,693,075, 6,762,286 and 6,806,063, U.S. Patent Publication Nos.20030100506 and 20040220106 and International Patent Publication Nos.WO1988010295, WO200183725, WO2003093465, WO200338162, WO2004083361,WO2004108763, WO2004029090, WO2004029091, WO2004111242 and WO2005123916.

As used herein, “modified factor VII polypeptides” and “modified factorVII” refer to a FVII polypeptide that has one or more amino aciddifferences compared to an unmodified factor VII polypeptide. The one ormore amino acid differences can be amino acid mutations such as one ormore amino acid replacements (substitutions), insertions or deletions,or can be insertions or deletions of entire domains, and anycombinations thereof. Typically, a modified FVII polypeptide has one ormore modifications in primary sequence compared to an unmodified FVIIpolypeptide. For example, a modified FVII polypeptide provided hereincan have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 30, 40, 50 or more amino acid differences compared to anunmodified FVII polypeptide. Any modification is contemplated as long asthe resulting polypeptide exhibits at least one FVII activity associatedwith a native FVII polypeptide, such as, for example, catalyticactivity, proteolytic activity, the ability to bind TF or the ability tobind activated platelets.

As used herein, “inhibitors of coagulation” refer to proteins ormolecules that act to inhibit or prevent coagulation or clot formation.The inhibition or prevention of coagulation can be observed in vivo orin vitro, and can be assayed using any method known in the artincluding, but not limited to, prothromboplastin time (PT) assay or theactivated partial thromboplastin time (aPTT) assay.

As used herein, tissue factor pathway inhibitor (TFPI, also referred toas TFPI-1) is a Kunitz-type inhibitor that is involved in the formationof a quaternary TF/FVIIa/TFPI/FXa inhibitory complex in which theactivity of FVIIa is inhibited. TFPI is expressed as two differentprecursor forms following alternative splicing, TFPIα (SEQ ID NO:75) andTFPIβ (SEQ ID NO:77) precursors, which are cleaved during secretion togenerate a 276 amino acid (SEQ ID NO:76) and a 223 amino acid (SEQ IDNO:78) mature protein, respectively. TFPI contains 3 Kunitz domains, ofwhich the Kunitz-1 domain is responsible for binding and inhibition ofFVIIa.

As used herein, TFPI-2 (also is known as placental protein 5 (PP5) andmatrix-associated serine protease inhibitor (MSPI)) refers to a homologof TFPI. The 213 amino acid mature TFPI-2 protein (SEQ ID NO:79)contains three Kunitz-type domains that exhibit 43%, 35% and 53% primarysequence identity with TFPI-1 Kunitz-type domains 1, 2, and 3,respectively. TFPI-2 plays a role in the regulation of extracellularmatrix digestion and remodeling, and is not thought to be an importantfactor in the coagulation pathway.

As used herein, antithrombin III (AT-III) is a serine protease inhibitor(serpin). AT-III is synthesized as a precursor protein containing 464amino acid residues (SEQ ID NO:95) that is cleaved during secretion torelease a 432 amino acid mature antithrombin (SEQ ID NO:96).

As used herein, cofactors refer to proteins or molecules that bind toother specific proteins or molecules to form an active complex. In someexamples, binding to a cofactor is required for optimal proteolyticactivity. For example, tissue factor (TF) is a cofactor of FVIIa.Binding of FVIIa to TF induces conformational changes that result inincreased proteolytic activity of FVIIa for its substrates, FX and FIX.

As used herein, tissue factor (TF) refers to a 263 amino acidstransmembrane glycoprotein (SEQ ID NO:97) that functions as a cofactorfor FVIIa. It is constitutively expressed by smooth muscle cells andfibroblasts, and helps to initiate coagulation by binding FVII and FVIIawhen these cells come in contact with the bloodstream following tissueinjury.

As used herein, activated platelet refers to a platelet that has beentriggered by the binding of molecules such as collagen, thromboxane A2,ADP and thrombin to undergo various changes in morphology, phenotype andfunction that ultimately promote coagulation. For example, an activatedplatelet changes in shape to a more amorphous form with projectingfingers. Activated platelets also undergo a “flip” of the cell membranesuch that phosphatidylserine and other negatively charged phospholipidsthat are normally present in the inner leaflet of the cell membrane aretranslocated to the outer, plasma-oriented surface. These membranes ofthe activated platelets provide the surface on which many of thereactions of the coagulation cascade are effected. Activated plateletsalso secrete vesicles containing such pro-coagulant factors as vWF, FV,thrombin, ADP and thromboxane A2, and adhere to one another to form aplatelet plug which is stabilized by fibrin to become a clot.

As used herein, increased binding and/or affinity for activatedplatelets, and any grammatical variations thereof, refers to an enhancedability of a polypeptide or protein, for example a FVII polypeptide, tobind to the surface of an activated platelet, as compared with areference polypeptide or protein. For example, the ability of a modifiedFVII polypeptide to bind to activated platelets can be greater than theability of the unmodified FVII polypeptide to bind to activatedplatelets. The binding and/or affinity of a polypeptide for activatedplatelets can be increased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%,400%, 500%, or more compared to the binding and/or affinity of anunmodified polypeptide. Assays to determine the binding and/or affinityof a polypeptide for activated platelets are known in the art. Bindingof a FVII polypeptide to activated platelets is mediated through theinteraction of amino acids in the Gla domain of the FVII polypeptide andnegatively charged phospholipids, such as phosphatidylserine, on theactivated platelet. As such, methods to assay for binding ofpolypeptides, such as FVII polypeptides, to activated platelets usemembranes and vesicles that contain phospholipids, such asphosphatidylserine. For example, the ability of a polypeptide to bind toan activated platelet is reflected by the ability of the polypeptide tobind to phospholipid vesicles, which can be measured by light scatteringtechniques.

As used herein, increased binding and/or affinity for phospholipids, andany grammatical variations thereof, refers to an enhanced ability of apolypeptide or protein to bind to phospholipids as compared with areference polypeptide or protein. Phospholipids can include anyphospholipids, but particularly include phosphatidylserine. The bindingand/or affinity of a polypeptide for phospholipids can be increased byat least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more comparedto the binding and/or affinity of an unmodified polypeptide. Assays todetermine the affinity and/or binding of a polypeptide to phospholipidsare known in the art. For example, FVII polypeptide binding tophospholipid vesicles can be determined by relative light scattering at90° to the incident light. The intensity of the light scatter with thephospholipid vesicles alone and with phospholipid vesicles with FVII ismeasured to determine the dissociation constant. Surface plasmaresonance, such as on a BIAcore biosensor instrument, also can be usedto measure the affinity of FVII polypeptides for phospholipid membranes.

As used herein, increased resistance to inhibitors or “increasedresistance to AT-III” or “increased resistance to TFPI” refers to anyamount of decreased sensitivity of a polypeptide to the inhibitoryeffects of an inhibitor, such as AT-III or TFPI, compared with areference polypeptide, such as an unmodified FVII polypeptide. Increasedresistance to an inhibitor, such as AT-III, can be assayed by assessingthe binding of a modified FVII polypeptide to an inhibitor. Increasedresistance to an inhibitor, such as AT-III, also can be assayed bymeasuring the intrinsic activity or coagulant activity of a FVIIpolypeptide in the presence of AT-III. Assays to determine the bindingof a polypeptide to an inhibitor are known in the art. For covalentinhibitors, such as, for example, AT-III, a second order rate constantfor inhibition can be measured. For non-covalent inhibitors, such as,for example, TFPI, a k_(i) can be measured. In addition, surface plasmaresonance, such as on a BIAcore biosensor instrument, also can be usedto measure the binding of FVII polypeptides to AT-III or otherinhibitors. However, for covalent inhibitors such as AT-III, only anon-rate can be measured using BIAcore. Assays to determine theinhibitory effect of, for example, AT-III on FVII coagulant activity orintrinsic activity also are known in the art. For example, the abilityof a modified FVII polypeptide to cleave its substrate FX in thepresence or absence of AT-III can be measured, and the degree to whichAT-III inhibits the reaction determined. This can be compared to theability of an unmodified FVII polypeptide to cleave its substrate FX inthe presence or absence of AT-III. A modified polypeptide that exhibitsincreased resistance to an inhibitor exhibits, for example, an increaseof 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%,300%, 400%, 500%, or more resistance to the effects of an inhibitorcompared to an unmodified polypeptide.

As used herein, “increased resistance to inhibition by Zn²⁺,” “increasedresistance to the inhibitory effects of Zn²⁺” or “increased resistanceto Zn²⁺” refers to any amount of decreased sensitivity of a polypeptideto the inhibitory effects of Zn²⁺ compared with a reference polypeptide,such as an unmodified FVII polypeptide. Increased resistance to Zn²⁺ canbe assayed by, for example, measuring the intrinsic activity orcoagulant activity of a FVII polypeptide in the presence of Zn²⁺, suchas described in Example 11. Increased resistance to the inhibitoryeffects of Zn²⁺ can be the result of decreased binding to Zn²⁺.Decreased binding to Zn²⁺ can be assayed by measuring the amount ofbound Zn²⁺ per molecule of FVIIa or by measuring the affinity of Zn²⁺binding to FVIIa or by measuring an IC₅₀ for inhibition of a FVIIaactivity by zinc. A modified polypeptide that exhibits increasedresistance to the inhibitory effects of Zn²⁺ exhibits, for example, anincrease of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%,200%, 300%, 400%, 500%, or more resistance to the effects of Zn²⁺compared to an unmodified polypeptide.

As used herein, a serum albumin binding sequence refers to a sequence ofamino acid residues that can effect binding to serum albumin. Thus, wheninserted into a FVII polypeptide, the serum albumin binding sequence canenhance the affinity for or binding to serum albumin of the FVIIpolypeptide. The ability of the modified FVII polypeptide containing theserum albumin binding sequence can therefore exhibit increased bindingand/or affinity for serum albumin. A modified polypeptide that exhibitsincreased binding and/or affinity for serum albumin exhibits, forexample, an increase of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100%, 200%, 300%, 400%, 500%, or more compared to the bindingand/or affinity of an unmodified polypeptide. Typically, serum albuminbinding sequences contain at least 10 or more amino acids, typically 10,11, 12, 13, 14, 15, 20, 30, 40 or more amino acids. Exemplary of serumalbumin binding sequences are those set forth in SEQ ID NOS:103-109.

As used herein, a platelet integrin α_(IIb)β₃ binding sequence refers toa sequence of amino acid residues that can effect binding to plateletintegrin α_(IIb)β₃. Thus, when inserted into a FVII polypeptide, theplatelet integrin α_(IIb)β₃ binding sequence can enhance the ability ofthe FVII polypeptide to bind to platelet integrin α_(IIb)β₃ and,therefore, platelets, including activated platelets. The ability of themodified FVII polypeptide containing the platelet integrin α_(IIb)β₃binding sequence can therefore exhibit increased binding and/or affinityfor platelet integrin α_(IIb)β₃ and/or platelets. A modified polypeptidethat exhibits increased binding and/or affinity for platelet integrinα_(IIb)β₃ exhibits, for example, an increase of 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or morecompared to the binding and/or affinity of an unmodified polypeptide.Typically, platelet integrin α_(IIb)β₃ binding sequences contain atleast 5 or more amino acids, typically 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 20, 30, 40 or more amino acids. Exemplary of platelet integrinα_(IIb)β₃ binding sequences are those set forth in SEQ ID NOS:110-112.

As used herein, a glycosylation site refers to an amino position in apolypeptide to which a carbohydrate moiety can be attached. Typically, aglycosylated protein contains one or more amino acid residues, such asasparagine or serine, for the attachment of the carbohydrate moieties.

As used herein, a native glycosylation site refers to an amino positionto which a carbohydrate moiety is attached in a wild-type polypeptide.There are four native glycosylation sites in FVII; two N-glycosylationsites at N145 and N322, and two O-glycosylation sites at S52 and S60,corresponding to amino acid positions in the mature FVII polypeptide setforth in SEQ ID NO:3.

As used herein, a non-native glycosylation site refers to an aminoposition to which a carbohydrate moiety is attached in a modifiedpolypeptide that is not present in a wild-type polypeptide. Non-nativeglycosylation sites can be introduced into a FVII polypeptide by aminoacid replacement. O-glycosylation sites can be created, for example, byamino acid replacement of a native residue with a serine or threonine.N-glycosylation sites can be created, for example, by establishing themotif Asn-Xaa-Ser/Thr/Cys, where Xaa is not proline. Creation of thisconsensus sequence by amino acid modification can involve, for example,a single amino acid replacement of a native amino acid residue with anasparagine, a single amino acid replacement of a native amino acidresidue with a serine, threonine or cysteine, or a double amino acidreplacement involving a first amino acid replacement of a native residuewith an asparagine and a second amino acid replacement of native residuewith a serine, threonine or cysteine.

As used herein, “biological activity” refers to the in vivo activitiesof a compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Biologicalactivities can be observed in in vitro systems designed to test or usesuch activities. Thus, for purposes herein a biological activity of aFVII polypeptide encompasses the coagulant activity.

As used herein the term “assess”, and grammatical variations thereof, isintended to include quantitative and qualitative determination in thesense of obtaining an absolute value for the activity of a polypeptide,and also of obtaining an index, ratio, percentage, visual or other valueindicative of the level of the activity. Assessment can be direct orindirect. For example, detection of cleavage of a substrate by apolypeptide can be by direct measurement of the product, or can beindirectly measured by determining the resulting activity of the cleavedsubstrate.

As used herein, “chymotrypsin numbering” refers to the amino acidnumbering of a mature chymotrypsin polypeptide of SEQ ID NO:80.Alignment of a protease domain of another protease, such as for examplethe protease domain of factor VII, can be made with chymotrypsin. Insuch an instance, the amino acids of factor VII that correspond to aminoacids of chymotrypsin are given the numbering of the chymotrypsin aminoacids. Corresponding positions can be determined by such alignment byone of skill in the art using manual alignments or by using the numerousalignment programs available (for example, BLASTP). Correspondingpositions also can be based on structural alignments, for example byusing computer simulated alignments of protein structure. Recitationthat amino acids of a polypeptide correspond to amino acids in adisclosed sequence refers to amino acids identified upon alignment ofthe polypeptide with the disclosed sequence to maximize identity orhomology (where conserved amino acids are aligned) using a standardalignment algorithm, such as the GAP algorithm. The correspondingchymotrypsin numbers of amino acid positions 1 to 406 of the FVIIpolypeptide set forth in SEQ ID NO:3 are provided in Table 1. The aminoacid positions relative to the sequence set forth in SEQ ID NO:3 are innormal font, the amino acid residues at those positions are in bold, andthe corresponding chymotrypsin numbers are in italics. For example, uponalignment of the mature factor VII (SEQ ID NO:3) with maturechymotrypsin (SEQ ID NO:80), the isoleucine (I) at amino acid position153 in factor VII is given the chymotrypsin numbering of I16. Subsequentamino acids are numbered accordingly. In one example, a glutamic acid(E) at amino acid position 210 of the mature factor VII (SEQ ID NO:3)corresponds to amino acid position E70 based on chymotrypsin numbering.Where a residue exists in a protease, but is not present inchymotrypsin, the amino acid residue is given a letter notation. Forexample, residues in chymotrypsin that are part of a loop with aminoacid 60 based on chymotrypsin numbering, but are inserted in the factorVII sequence compared to chymotrypsin, are referred to for example asK60a, 160b, K60c or N60d. These residues correspond to K197, I198, K199and N200, respectively, by numbering relative to the mature factor VIIsequence (SEQ ID NO:3).

TABLE 1 Chymotryspin numbering of factor VII 153 154 155 156 157 158 159160 161 162 163 164 165 166 167 I V G G K V C P K G E C P W Q  16  17 18  19  20  21  22  23  24  25  26  27  28  29  30 168 169 170 171 172173 174 175 176 177 178 179 180 181 182 V L L L V N G A Q L C G G T L 31  32  33  34  35  37  38  39  40  41  42  43  44  45  46 183 184 185186 187 188 189 190 191 192 193 194 195 196 197 I N T I W V V S A A H CF D K  47  48  49  50  51  52  53  54  55  56  57  58  59  60  60A 198199 200 201 202 203 204 205 206 207 208 209 210 211 212 I K N W R N L IA V L G E H D  60B  60C  60D  61  62  63  64  65  66  67  68  69  70  71 72 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 L S E HD G D E Q S R R V A Q  73  74  75  76  77  78  79  80  81  82  83  84 85  86  87 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242V I I P S T Y V P G T T N H D  88  89  90  91  92  93  94  95  96  97 98  99 100 101 102 243 244 245 246 247 248 249 250 251 252 253 254 255256 257 I A L L R L H Q P V V L T D H 103 104 105 106 107 108 109 110111 112 113 114 115 116 117 258 259 260 261 262 263 264 265 266 267 268269 270 271 272 V V P L C L P E R T F S E R T 118 119 120 121 122 123124 125 126 127 128 129 129A 129B 129C 273 274 275 276 277 278 279 280281 282 283 284 285 286 287 L A F V R F S L V S G W G Q L 129D 129E 129F129G 134 135 136 137 138 139 140 141 142 143 144 288 289 290 291 292 293294 295 296 297 298 299 300 301 302 L D R G A T A L E L M V L N V 145146 147 149 150 151 152 153 154 155 156 157 158 159 160 303 304 305 306307 308 309 310 311 312 313 314 315 316 317 P R L M T Q D C L Q Q S R KV 161 162 163 164 165 166 167 168 169 170 170A 170B 170C 170D 170E 318319 320 321 322 323 324 325 326 327 328 329 330 331 332 G D S P N I T EY M F C A G Y 170F 170G 170H 170I 175 176 177 178 179 180 181 182 183184A 184 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 S DG S K D S C K G D S G G P 185 186 187 188A 188 189 190 191 192 193 194195 196 197 198 348 349 350 351 352 353 354 355 356 357 358 359 360 361362 H A T H Y R G T W Y L T G I V 199 200 201 202 203 204 205 206 207208 209 210 211 212 213 363 364 365 366 367 368 369 370 371 372 373 374375 376 377 S W G Q G C A T V G H F G V Y 214 215 216 217 219 220 221A221 222 223 224 225 226 227 228 378 379 380 381 382 383 384 385 386 387388 389 390 391 392 T R V S Q Y I E W L Q K L M R 229 230 231 232 233234 235 236 237 238 239 240 241 242 243 393 394 395 396 397 398 399 400401 402 403 404 405 406 S E P R P G V L L R A P F P 244 245 246 247 248249 250 251 252 253 254 255 256 257

As used herein, nucleic acids include DNA, RNA and analogs thereof,including peptide nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double-stranded. When referring to probes orprimers, which are optionally labeled, such as with a detectable label,such as a fluorescent or radiolabel, single-stranded molecules arecontemplated. Such molecules are typically of a length such that theirtarget is statistically unique or of low copy number (typically lessthan 5, generally less than 3) for probing or priming a library.Generally a probe or primer contains at least 14, 16 or 30 contiguousnucleotides of sequence complementary to or identical to a gene ofinterest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleicacids long.

As used herein, a peptide refers to a polypeptide that is from 2 to 40amino acids in length.

As used herein, the amino acids that occur in the various sequences ofamino acids provided herein are identified according to their known,three-letter or one-letter abbreviations (Table 2). The nucleotideswhich occur in the various nucleic acid fragments are designated withthe standard single-letter designations used routinely in the art.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids include the twentynaturally-occurring amino acids, non-natural amino acids and amino acidanalogs (i.e., amino acids wherein the α-carbon has a side chain).

In keeping with standard polypeptide nomenclature described in J. Biol.Chem., 243: 3557-3559 (1968), and adopted 37 C.F.R. §§1.821-1.822,abbreviations for the amino acid residues are shown in Table 2:

TABLE 2 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys Cysteine XXaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by formulae have a left to right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus. In addition, thephrase “amino acid residue” is broadly defined to include the aminoacids listed in the Table of Correspondence (Table 2) and modified andunusual amino acids, such as those referred to in 37 C.F.R.§§1.821-1.822, and incorporated herein by reference. Furthermore, itshould be noted that a dash at the beginning or end of an amino acidresidue sequence indicates a peptide bond to a further sequence of oneor more amino acid residues, to an amino-terminal group such as NH₂ orto a carboxyl-terminal group such as COOH.

As used herein, a “hydrophobic amino acid” includes any one of the aminoacids determined to be hydrophobic using the Eisenberg hydrophobicityconsensus scale. Exemplary are the naturally occurring hydrophobic aminoacids, such as isoleucine, phenylalanine, valine, leucine, tryptophan,methionine, alanine, glycine, cysteine and tyrosine (Eisenberg et al.,(1982) Faraday Symp. Chem. Soc. 17:109-120). Non-naturally-occurringhydrophobic amino acids also are included.

As used herein, an “acidic amino acid” includes among thenaturally-occurring amino acids aspartic acid and glutamic acidresidues. Non-naturally-occurring acidic amino acids also are included.

As used herein, “naturally occurring amino acids” refer to the 20L-amino acids that occur in polypeptides.

As used herein, “non-natural amino acid” refers to an organic compoundcontaining an amino group and a carboxylic acid group that is not one ofthe naturally-occurring amino acids listed in Table 2. Non-naturallyoccurring amino acids thus include, for example, amino acids or analogsof amino acids other than the 20 naturally-occurring amino acids andinclude, but are not limited to, the D-isostereomers of amino acids.Exemplary non-natural amino acids are known to those of skill in the artand can be included in a modified factor VII polypeptide.

As used herein, a DNA construct is a single or double stranded, linearor circular DNA molecule that contains segments of DNA combined andjuxtaposed in a manner not found in nature. DNA constructs exist as aresult of human manipulation, and include clones and other copies ofmanipulated molecules.

As used herein, a DNA segment is a portion of a larger DNA moleculehaving specified attributes. For example, a DNA segment encoding aspecified polypeptide is a portion of a longer DNA molecule, such as aplasmid or plasmid fragment, which, when read from the 5′ to 3′direction, encodes the sequence of amino acids of the specifiedpolypeptide.

As used herein, the term polynucleotide means a single- ordouble-stranded polymer of deoxyribonucleotides or ribonucleotide basesread from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, andcan be isolated from natural sources, synthesized in vitro, or preparedfrom a combination of natural and synthetic molecules. The length of apolynucleotide molecule is given herein in terms of nucleotides(abbreviated “nt”) or base pairs (abbreviated “bp”). The termnucleotides is used for single- and double-stranded molecules where thecontext permits. When the term is applied to double-stranded moleculesit is used to denote overall length and will be understood to beequivalent to the term base pairs. It will be recognized by thoseskilled in the art that the two strands of a double-strandedpolynucleotide can differ slightly in length and that the ends thereofcan be staggered; thus all nucleotides within a double-strandedpolynucleotide molecule can not be paired. Such unpaired ends will, ingeneral, not exceed 20 nucleotides in length.

As used herein, “primary sequence” refers to the sequence of amino acidresidues in a polypeptide.

As used herein, “similarity” between two proteins or nucleic acidsrefers to the relatedness between the sequence of amino acids of theproteins or the nucleotide sequences of the nucleic acids. Similaritycan be based on the degree of identity and/or homology of sequences ofresidues and the residues contained therein. Methods for assessing thedegree of similarity between proteins or nucleic acids are known tothose of skill in the art. For example, in one method of assessingsequence similarity, two amino acid or nucleotide sequences are alignedin a manner that yields a maximal level of identity between thesequences. “Identity” refers to the extent to which the amino acid ornucleotide sequences are invariant. Alignment of amino acid sequences,and to some extent nucleotide sequences, also can take into accountconservative differences and/or frequent substitutions in amino acids(or nucleotides). Conservative differences are those that preserve thephysico-chemical properties of the residues involved. Alignments can beglobal (alignment of the compared sequences over the entire length ofthe sequences and including all residues) or local (the alignment of aportion of the sequences that includes only the most similar region orregions).

As used herein, the terms “homology” and “identity” are usedinterchangeably, but homology for proteins can include conservativeamino acid changes. In general to identify corresponding positions thesequences of amino acids are aligned so that the highest order match isobtained (see, e.g.: Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; Carillo et al. (1988) SIAM J Applied Math 48:1073).

As use herein, “sequence identity” refers to the number of identicalamino acids (or nucleotide bases) in a comparison between a test and areference polypeptide or polynucleotide. Homologous polypeptides referto a pre-determined number of identical or homologous amino acidresidues. Homology includes conservative amino acid substitutions aswell identical residues. Sequence identity can be determined by standardalignment algorithm programs used with default gap penalties establishedby each supplier. Homologous nucleic acid molecules refer to apre-determined number of identical or homologous nucleotides. Homologyincludes substitutions that do not change the encoded amino acid (i.e.,“silent substitutions”) as well identical residues. Substantiallyhomologous nucleic acid molecules hybridize typically at moderatestringency or at high stringency all along the length of the nucleicacid or along at least about 70%, 80% or 90% of the full-length nucleicacid molecule of interest. Also contemplated are nucleic acid moleculesthat contain degenerate codons in place of codons in the hybridizingnucleic acid molecule. (For determination of homology of proteins,conservative amino acids can be aligned as well as identical aminoacids; in this case, percentage of identity and percentage homologyvaries). Whether any two nucleic acid molecules have nucleotidesequences (or any two polypeptides have amino acid sequences) that areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can bedetermined using known computer algorithms such as the “FAST A” program,using for example, the default parameters as in Pearson et al. Proc.Natl. Acad. Sci. USA 85: 2444 (1988) (other programs include the GCGprogram package (Devereux, J., et al., Nucleic Acids Research 12(I): 387(1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J. Molec. Biol.215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego (1994), and Carillo et al. SIAM J Applied Math 48: 1073(1988)). For example, the BLAST function of the National Center forBiotechnology Information database can be used to determine identity.Other commercially or publicly available programs include DNAStar“MegAlign” program (Madison, Wis.) and the University of WisconsinGenetics Computer Group (UWG) “Gap” program (Madison Wis.)). Percenthomology or identity of proteins and/or nucleic acid molecules can bedetermined, for example, by comparing sequence information using a GAPcomputer program (e.g., Needleman et al. J. Mol. Biol. 48: 443 (1970),as revised by Smith and Waterman (Adv. Appl. Math. 2: 482 (1981)).Briefly, a GAP program defines similarity as the number of alignedsymbols (i.e., nucleotides or amino acids) that are similar, divided bythe total number of symbols in the shorter of the two sequences. Defaultparameters for the GAP program can include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non identities)and the weighted comparison matrix of Gribskov et al. Nucl. Acids Res.14: 6745 (1986), as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

Therefore, as used herein, the term “identity” represents a comparisonbetween a test and a reference polypeptide or polynucleotide. In onenon-limiting example, “at least 90% identical to” refers to percentidentities from 90 to 100% relative to the reference polypeptides.Identity at a level of 90% or more is indicative of the fact that,assuming for exemplification purposes a test and referencepolynucleotide length of 100 amino acids are compared, no more than 10%(i.e., 10 out of 100) of amino acids in the test polypeptide differsfrom that of the reference polypeptides. Similar comparisons can be madebetween a test and reference polynucleotides. Such differences can berepresented as point mutations randomly distributed over the entirelength of an amino acid sequence or they can be clustered in one or morelocations of varying length up to the maximum allowable, e.g., 10/100amino acid difference (approximately 90% identity). Differences aredefined as nucleic acid or amino acid substitutions, insertions ordeletions. At the level of homologies or identities above about 85-90%,the result should be independent of the program and gap parameters set;such high levels of identity can be assessed readily, often withoutrelying on software.

As used herein, it also is understood that the terms “substantiallyidentical” or “similar” varies with the context as understood by thoseskilled in the relevant art, but that those of skill can assess such.

As used herein, an aligned sequence refers to the use of homology(similarity and/or identity) to align corresponding positions in asequence of nucleotides or amino acids. Typically, two or more sequencesthat are related by 50% or more identity are aligned. An aligned set ofsequences refers to 2 or more sequences that are aligned atcorresponding positions and can include aligning sequences derived fromRNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.

As used herein, “specifically hybridizes” refers to annealing, bycomplementary base-pairing, of a nucleic acid molecule (e.g. anoligonucleotide) to a target nucleic acid molecule. Those of skill inthe art are familiar with in vitro and in vivo parameters that affectspecific hybridization, such as length and composition of the particularmolecule. Parameters particularly relevant to in vitro hybridizationfurther include annealing and washing temperature, buffer compositionand salt concentration. Exemplary washing conditions for removingnon-specifically bound nucleic acid molecules at high stringency are0.1×SSPE, 0.1% SDS, 65° C., and at medium stringency are 0.2×SSPE, 0.1%SDS, 50° C. Equivalent stringency conditions are known in the art. Theskilled person can readily adjust these parameters to achieve specifichybridization of a nucleic acid molecule to a target nucleic acidmolecule appropriate for a particular application.

As used herein, isolated or purified polypeptide or protein orbiologically-active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell of tissue fromwhich the protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. Preparationscan be determined to be substantially free if they appear free ofreadily detectable impurities as determined by standard methods ofanalysis, such as thin layer chromatography (TLC), gel electrophoresisand high performance liquid chromatography (HPLC), used by those ofskill in the art to assess such purity, or sufficiently pure such thatfurther purification would not detectably alter the physical andchemical properties, such as proteolytic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound, however, can be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

The term substantially free of cellular material includes preparationsof proteins in which the protein is separated from cellular componentsof the cells from which it is isolated or recombinantly-produced. In oneembodiment, the term substantially free of cellular material includespreparations of protease proteins having less that about 30% (by dryweight) of non-protease proteins (also referred to herein as acontaminating protein), generally less than about 20% of non-proteaseproteins or 10% of non-protease proteins or less that about 5% ofnon-protease proteins. When the protease protein or active portionthereof is recombinantly produced, it also is substantially free ofculture medium, i.e., culture medium represents less than, about, orequal to 20%, 10% or 5% of the volume of the protease proteinpreparation.

As used herein, the term substantially free of chemical precursors orother chemicals includes preparations of protease proteins in which theprotein is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. The term includespreparations of protease proteins having less than about 30% (by dryweight), 20%, 10%, 5% or less of chemical precursors or non-proteasechemicals or components.

As used herein, production by recombinant methods by using recombinantDNA methods refers to the use of the well known methods of molecularbiology for expressing proteins encoded by cloned DNA.

As used herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous nucleic acid into cells for eitherexpression or replication thereof. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Also contemplated are vectorsthat are artificial chromosomes, such as bacterial artificialchromosomes, yeast artificial chromosomes and mammalian artificialchromosomes. Selection and use of such vehicles are well known to thoseof skill in the art.

As used herein, expression refers to the process by which nucleic acidis transcribed into mRNA and translated into peptides, polypeptides, orproteins. If the nucleic acid is derived from genomic DNA, expressioncan, if an appropriate eukaryotic host cell or organism is selected,include processing, such as splicing of the mRNA.

As used herein, an expression vector includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or can contain elements of both.Thus, an expression vector refers to a recombinant DNA or RNA construct,such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome.

As used herein, vector also includes “virus vectors” or “viral vectors.”Viral vectors are engineered viruses that are operatively linked toexogenous genes to transfer (as vehicles or shuttles) the exogenousgenes into cells.

As used herein, an adenovirus refers to any of a group of DNA-containingviruses that cause conjunctivitis and upper respiratory tract infectionsin humans.

As used herein, naked DNA refers to histone-free DNA that can be usedfor vaccines and gene therapy. Naked DNA is the genetic material that ispassed from cell to cell during a gene transfer processed calledtransformation or transfection. In transformation or transfection,purified or naked DNA that is taken up by the recipient cell will givethe recipient cell a new characteristic or phenotype.

As used herein, operably or operatively linked when referring to DNAsegments means that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiates inthe promoter and proceeds through the coding segment to the terminator.

As used herein, an agent that modulates the activity of a protein orexpression of a gene or nucleic acid either decreases or increases orotherwise alters the activity of the protein or, in some manner, up- ordown-regulates or otherwise alters expression of the nucleic acid in acell.

As used herein, a “chimeric protein” or “fusion protein” refers to apolypeptide operatively-linked to a different polypeptide. A chimeric orfusion protein provided herein can include one or more FVIIpolypeptides, or a portion thereof, and one or more other polypeptidesfor any one or more of a transcriptional/translational control signals,signal sequences, a tag for localization, a tag for purification, partof a domain of an immunoglobulin G, and/or a targeting agent. A chimericFVII polypeptide also includes those having their endogenous domains orregions of the polypeptide exchanged with another polypeptide. Thesechimeric or fusion proteins include those produced by recombinant meansas fusion proteins, those produced by chemical means, such as bychemical coupling, through, for example, coupling to sulfhydryl groups,and those produced by any other method whereby at least one polypeptide(i.e. FVII), or a portion thereof, is linked, directly or indirectly vialinker(s) to another polypeptide.

As used herein, operatively-linked when referring to a fusion proteinrefers to a protease polypeptide and a non-protease polypeptide that arefused in-frame to one another. The non-protease polypeptide can be fusedto the N-terminus or C-terminus of the protease polypeptide.

As used herein, a targeting agent, is any moiety, such as a protein oreffective portion thereof, that provides specific binding to a cellsurface molecule, such a cell surface receptor, which in some instancescan internalize a bound conjugate or portion thereof. A targeting agentalso can be one that promotes or facilitates, for example, affinityisolation or purification of the conjugate; attachment of the conjugateto a surface; or detection of the conjugate or complexes containing theconjugate.

As used herein, derivative or analog of a molecule refers to a portionderived from or a modified version of the molecule.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions, andcharacterized by identifiable symptoms. Diseases and disorders ofinterest herein are those involving coagulation, including thosemediated by coagulation proteins and those in which coagulation proteinsplay a role in the etiology or pathology. Diseases and disorders alsoinclude those that are caused by the absence of a protein such as inhemophilia, and of particular interest herein are those disorders wherecoagulation does not occur due to a deficiency of defect in acoagulation protein.

As used herein, “procoagulant” refers to any substance that promotesblood coagulation.

As used herein, “anticoagulant” refers to any substance that inhibitsblood coagulation

As used herein, “hemophilia” refers to a bleeding disorder caused by adeficiency in a blood clotting factors. Hemophilia can be the result,for example, of absence, reduced expression, or reduced function of aclotting factor. The most common type of hemophilia is hemophilia A,which results from a deficiency in factor VIII. The second most commontype of hemophilia is hemophilia B, which results from a deficiency infactor IX. Hemophilia C, also called FXI deficiency, is a milder andless common form of hemophila.

As used herein, “congenital hemophilia” refers to types of hemophiliathat are inherited. Congenital hemophilia results from mutation,deletion, insertion, or other modification of a clotting factor gene inwhich the production of the clotting factor is absent, reduced, ornon-functional. For example, hereditary mutations in clotting factorgenes, such as factor VIII and factor IX result in the congenitalhemophilias, Hemophilia A and B, respectively.

As used herein, “acquired hemophilia” refers to a type of hemophiliathat develops in adulthood from the production of autoantibodies thatinactivate FVIII.

As used herein, “bleeding disorder” refers to a condition in which thesubject has a decreased ability to control bleeding. Bleeding disorderscan be inherited or acquired, and can result from, for example, defectsor deficiencies in the coagulation pathway, defects or deficiencies inplatelet activity, or vascular defects.

As used herein, “acquired bleeding disorder” refers to bleedingdisorders that results from clotting deficiencies caused by conditionssuch as liver disease, vitamin K deficiency, or coumadin (warfarin) orother anti-coagulant therapy.

As used herein, “treating” a subject having a disease or condition meansthat a polypeptide, composition or other product provided herein isadministered to the subject.

As used herein, a therapeutic agent, therapeutic regimen,radioprotectant, or chemotherapeutic mean conventional drugs and drugtherapies, including vaccines, which are known to those skilled in theart. Radiotherapeutic agents are well known in the art.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered. Hence treatment encompasses prophylaxis, therapy and/or cure.Treatment also encompasses any pharmaceutical use of the compositionsherein. Treatment also encompasses any pharmaceutical use of a modifiedFVII and compositions provided herein.

As used herein, amelioration of the symptoms of a particular disease ordisorder by a treatment, such as by administration of a pharmaceuticalcomposition or other therapeutic, refers to any lessening, whetherpermanent or temporary, lasting or transient, of the symptoms that canbe attributed to or associated with administration of the composition ortherapeutic.

As used herein, prevention or prophylaxis refers to methods in which therisk of developing disease or condition is reduced. Prophylaxis includesreduction in the risk of developing a disease or condition and/or aprevention of worsening of symptoms or progression of a disease orreduction in the risk of worsening of symptoms or progression of adisease.

As used herein an effective amount of a compound or composition fortreating a particular disease is an amount that is sufficient toameliorate, or in some manner reduce the symptoms associated with thedisease. Such amount can be administered as a single dosage or can beadministered according to a regimen, whereby it is effective. The amountcan cure the disease but, typically, is administered in order toameliorate the symptoms of the disease. Typically, repeatedadministration is required to achieve a desired amelioration ofsymptoms.

As used herein, “therapeutically effective amount” or “therapeuticallyeffective dose” refers to an agent, compound, material, or compositioncontaining a compound that is at least sufficient to produce atherapeutic effect. An effective amount is the quantity of a therapeuticagent necessary for preventing, curing, ameliorating, arresting orpartially arresting a symptom of a disease or disorder.

As used herein, “patient” or “subject” to be treated includes humans andor non-human animals, including mammals. Mammals include primates, suchas humans, chimpanzees, gorillas and monkeys; domesticated animals, suchas dogs, horses, cats, pigs, goats, cows; and rodents such as mice,rats, hamsters and gerbils.

As used herein, a combination refers to any association between two oramong more items. The association can be spatial or refer to the use ofthe two or more items for a common purpose.

As used herein, a composition refers to any mixture of two or moreproducts or compounds (e.g., agents, modulators, regulators, etc.). Itcan be a solution, a suspension, liquid, powder, a paste, aqueous ornon-aqueous formulations or any combination thereof.

As used herein, an “article of manufacture” is a product that is madeand sold. As used throughout this application, the term is intended toencompass modified protease polypeptides and nucleic acids contained inarticles of packaging.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, a “kit” refers to a packaged combination, optionallyincluding reagents and other products and/or components for practicingmethods using the elements of the combination. For example, kitscontaining a modified protease polypeptide or nucleic acid moleculeprovided herein and another item for a purpose including, but notlimited to, administration, diagnosis, and assessment of a biologicalactivity or property are provided. Kits optionally include instructionsfor use.

As used herein, antibody includes antibody fragments, such as Fabfragments, which are composed of a light chain and the variable regionof a heavy chain.

As used herein, a receptor refers to a molecule that has an affinity fora particular ligand. Receptors can be naturally-occurring or syntheticmolecules. Receptors also can be referred to in the art as anti-ligands.

As used herein, animal includes any animal, such as, but not limited to;primates including humans, gorillas and monkeys; rodents, such as miceand rats; fowl, such as chickens; ruminants, such as goats, cows, deer,sheep; ovine, such as pigs and other animals. Non-human animals excludehumans as the contemplated animal. The proteases provided herein arefrom any source, animal, plant, prokaryotic and fungal.

As used herein, gene therapy involves the transfer of heterologousnucleic acid, such as DNA, into certain cells, target cells, of amammal, particularly a human, with a disorder or condition for whichsuch therapy is sought. The nucleic acid, such as DNA, is introducedinto the selected target cells, such as directly or in a vector or otherdelivery vehicle, in a manner such that the heterologous nucleic acid,such as DNA, is expressed and a therapeutic product encoded thereby isproduced. Alternatively, the heterologous nucleic acid, such as DNA, canin some manner mediate expression of DNA that encodes the therapeuticproduct, or it can encode a product, such as a peptide or RNA that insome manner mediates, directly or indirectly, expression of atherapeutic product. Genetic therapy also can be used to deliver nucleicacid encoding a gene product that replaces a defective gene orsupplements a gene product produced by the mammal or the cell in whichit is introduced. The introduced nucleic acid can encode a therapeuticcompound, such as a protease or modified protease, that is not normallyproduced in the mammalian host or that is not produced intherapeutically effective amounts or at a therapeutically useful time.The heterologous nucleic acid, such as DNA, encoding the therapeuticproduct can be modified prior to introduction into the cells of theafflicted host in order to enhance or otherwise alter the product orexpression thereof. Genetic therapy also can involve delivery of aninhibitor or repressor or other modulator of gene expression.

As used herein, heterologous nucleic acid is nucleic acid that is notnormally produced in vivo by the cell in which it is expressed or thatis produced by the cell but is at a different locus or expresseddifferently or that mediates or encodes mediators that alter expressionof endogenous nucleic acid, such as DNA, by affecting transcription,translation, or other regulatable biochemical processes. Heterologousnucleic acid is generally not endogenous to the cell into which it isintroduced, but has been obtained from another cell or preparedsynthetically. Heterologous nucleic acid can be endogenous, but isnucleic acid that is expressed from a different locus or altered in itsexpression. Generally, although not necessarily, such nucleic acidencodes RNA and proteins that are not normally produced by the cell orin the same way in the cell in which it is expressed. Heterologousnucleic acid, such as DNA, also can be referred to as foreign nucleicacid, such as DNA. Thus, heterologous nucleic acid or foreign nucleicacid includes a nucleic acid molecule not present in the exactorientation or position as the counterpart nucleic acid molecule, suchas DNA, is found in a genome. It also can refer to a nucleic acidmolecule from another organism or species (i.e., exogenous).

Any nucleic acid, such as DNA, that one of skill in the art wouldrecognize or consider as heterologous or foreign to the cell in whichthe nucleic acid is expressed is herein encompassed by heterologousnucleic acid; heterologous nucleic acid includes exogenously addednucleic acid that also is expressed endogenously. Examples ofheterologous nucleic acid include, but are not limited to, nucleic acidthat encodes traceable marker proteins, such as a protein that confersdrug resistance, nucleic acid that encodes therapeutically effectivesubstances, such as anti-cancer agents, enzymes and hormones, andnucleic acid, such as DNA, that encodes other types of proteins, such asantibodies. Antibodies that are encoded by heterologous nucleic acid canbe secreted or expressed on the surface of the cell in which theheterologous nucleic acid has been introduced.

As used herein, a therapeutically effective product for gene therapy isa product that is encoded by heterologous nucleic acid, typically DNA,that, upon introduction of the nucleic acid into a host, a product isexpressed that ameliorates or eliminates the symptoms, manifestations ofan inherited or acquired disease or that cures the disease. Alsoincluded are biologically active nucleic acid molecules, such as RNAiand antisense.

As used herein, recitation that a polypeptide “consists essentially” ofa recited sequence of amino acids means that only the recited portion,or a fragment thereof, of the full-length polypeptide is present. Thepolypeptide can optionally, and generally will, include additional aminoacids from another source or can be inserted into another polypeptide

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to compound, comprising “an extracellular domain”includes compounds with one or a plurality of extracellular domains.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 bases” means “about 5 bases” and also “5 bases.”

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally substitutedgroup means that the group is unsubstituted or is substituted.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).

B. Hemostasis Overview

Provided herein are modified Factor VII (FVII) polypeptides. Such FVIIpolypeptides are designed to have increased coagulant activity.Accordingly, these polypeptides have a variety of uses and applications,for example, as therapeutics for modulating hemostasis, and otherrelated biological processes. To appreciate the modifications providedherein and the use of such modified FVII molecules, an understanding ofthe haemostatic system and the blood coagulation cascade isadvantageous. The following discussion provides such background,prefatory to a discussion of factor VII, and modifications thereof.

Hemostasis is the physiological mechanism that stems the bleeding thatresults from injury to the vasculature. Normal hemostasis depends oncellular components and soluble plasma proteins, and involves a seriesof signaling events that ultimately leads to the formation of a bloodclot. Coagulation is quickly initiated after an injury occurs to theblood vessel and endothelial cells are damaged. In the primary phase ofcoagulation, platelets are activated to form a haemostatic plug at thesite of injury. Secondary hemostasis follows involving plasmacoagulation factors, which act in a proteolytic cascade resulting in theformation of fibrin strands which strengthen the platelet plug.

Upon vessel injury, the blood flow to the immediate injured area isrestricted by vascular constriction allowing platelets to adhere to thenewly-exposed fibrillar collagen on the subendothelial connectivetissue. This adhesion is dependent upon the von Willebrand factor (vWF),which binds to the endothelium within three seconds of injury, therebyfacilitating platelet adhesion and aggregation. Activation of theaggregated platelets results in the secretion of a variety of factors,including ADP, ATP, thromboxane and serotonin. Adhesion molecules,fibrinogen, vWF, thrombospondin and fibronectin also are released. Suchsecretion promotes additional adhesion and aggregation of platelets,increased platelet activation and blood vessel constriction, andexposure of anionic phospholipids on the platelet surface that serve asplatforms for the assembly of blood coagulation enzyme complexes. Theplatelets change shape leading to pseudopodia formation, which furtherfacilitates aggregation to other platelets resulting in a loose plateletplug.

A clotting cascade of peptidases (the coagulation cascade) issimultaneously initiated. The coagulation cascade involves a series ofactivation events involving proteolytic cleavage. In such a cascade, aninactive protein of a serine protease (also called a zymogen) isconverted to an active protease by cleavage of one or more peptidebonds, which then serves as the activating protease for the next zymogenmolecule in the cascade, ultimately resulting in clot formation by thecross-linking of fibrin. For example, the cascade generates activatedmolecules such as thrombin (from cleavage of prothrombin), which furtheractivates platelets, and also generates fibrin from cleavage offibrinogen. Fibrin then forms a cross-linked polymer around the plateletplug to stabilize the clot. Upon repair of the injury, fibrin isdigested by the fibrinolytic system, the major components of which areplasminogen and tissue-type plasminogen activator (tPA). Both of theseproteins are incorporated into polymerizing fibrin, where they interactto generate plasmin, which, in turn, acts on fibrin to dissolve thepreformed clot. During clot formation, coagulation factor inhibitorsalso circulate through the blood to prevent clot formation beyond theinjury site.

The interaction of the system, from injury to clot formation andsubsequent fibrinolysis, is described below.

1. Platelet Adhesion and Aggregation

The clotting of blood is actively circumvented under normal conditions.The vascular endothelium supports vasodilation, inhibits plateletadhesion and activation, suppresses coagulation, enhances fibrincleavage and is anti-inflammatory in character. Vascular endothelialcells secrete molecules such as nitrous oxide (NO) and prostacylin,which inhibit platelet aggregation and dilate blood vessels. Release ofthese molecules activates soluble guanylate cyclases (sGC) andcGMP-dependent protein kinase I (cGKI) and increases cyclic guanosinemonophosphate (cGMP) levels, which cause relaxation of the smooth musclein the vessel wall. Furthermore, endothelial cells express cell-surfaceADPases, such as CD39, which control platelet activation and aggregationby converting ADP released from platelets into adenine nucleotideplatelet inhibitors. The endothelium also plays an important role in theregulation of the enzymes in the fibrinolytic cascade. Endothelial cellsdirectly promote the generation of plasmin through the expression ofreceptors of plasminogen (annexin II) and urokinase, as well as thesecretion of tissue-type and urokinase plasminogen activators, all ofwhich promote clot clearance. In a final layer of prothromboticregulation, endothelial cells play an active role in inhibiting thecoagulation cascade by producing heparan sulfate, which increases thekinetics of antithrombin III inhibition of thrombin and othercoagulation factors.

Under acute vascular trauma, however, vasoconstrictor mechanismspredominate and the endothelium becomes prothrombotic, procoagulatoryand proinflammatory in nature. This is achieved by a reduction ofendothelial dilating agents: adenosine, NO and prostacyclin; and thedirect action of ADP, serotonin and thromboxane on vascular smoothmuscle cells to elicit their contraction (Becker, Heindl et al. 2000).The chief trigger for the change in endothelial function that leads tothe formation of haemostatic thrombus is the loss of the endothelialcell barrier between blood and extracellular matrix (ECM) components(Ruggeri (2002) Nat Med 8:1227-1234). Circulating platelets identify anddiscriminate areas of endothelial lesions and adhere to the exposed subendothelium. Their interaction with the various thrombogenic substratesand locally-generated or released agonists results in plateletactivation. This process is described as possessing two stages, 1)adhesion: the initial tethering to a surface, and 2) aggregation: theplatelet-platelet cohesion (Savage et al. (2001) Curr Opin Hematol8:270-276).

Platelet adhesion is initiated when the circulating platelets bind toexposed collagen through interaction with collagen binding proteins onthe cell surface, and through interaction with vWF, also present on theendothelium. vWF protein is a multimeric structure of variable size,secreted in two directions by the endothelium; basolaterally and intothe bloodstream. vWF also binds to factor VIII, which is important inthe stabilization of factor VIII and its survival in the circulation.

Platelet adhesion and subsequent activation is achieved when vWF bindsvia its A1 domain to GPIb (part of the platelet glycoprotein receptorcomplex GPIb-IX-V). The interaction between vWF and GPIb is regulated byshear force such that an increase in the shear stress results in acorresponding increase in the affinity of vWF for GPIb. Integrin α1β2,also known on leukocytes as VLA-2, is the major collagen receptor onplatelets, and engagement through this receptor generates theintracellular signals that contribute to platelet activation. Bindingthrough α1β2 facilitates the engagement of the lower-affinity collagenreceptor, GP VI. This is part of the immunoglobulin superfamily and isthe receptor that generates the most potent intracellular signals forplatelet activation. Platelet activation results in the release ofadenosine diphosphate (ADP), which is converted to thromboxane A2.

Platelet activation also results in the surface expression of plateletglycoprotein IIb-IIIa (GP IIb-IIIa) receptors, also known as plateletintegrin α_(IIb)β₃. GP IIb-IIIa receptors allow the adherence ofplatelets to each other (i.e. aggregation) by virtue of fibrinogenmolecules linking the platelets through these receptors. This results inthe formation of a platelet plug at the site of injury to help preventfurther blood loss, while the damaged vascular tissue releases factorsthat initiate the coagulation cascade and the formation of a stabilizingfibrin mesh around the platelet plug.

2. Coagulation Cascade

The coagulation pathway is a proteolytic pathway where each enzyme ispresent in the plasma as a zymogen, or inactive form. Cleavage of thezymogen is regulated to release the active form from the precursormolecule. Cofactors of the activated proteases, such as theglycoproteins FVIII and FV, also are activated in the cascade reactionand play a role in clot formation. The pathway functions as a series ofpositive and negative feedback loops which control the activationprocess, where the ultimate goal is to produce thrombin, which can thenconvert soluble fibrinogen into fibrin to form a clot. The factors inthe coagulation are typically given a roman numeral number, with a lowercase “a” appended to indicate an activated form. Table 3 below setsforth an exemplary list of the factors, including their common name, andtheir role in the coagulation cascade. Generally, these proteinsparticipate in blood coagulation through one or more of the intrinsic,extrinsic or common pathway of coagulation (see FIG. 1). As discussedbelow, these pathways are interconnected, and blood coagulation isbelieved to occur through a cell-based model of activation with FactorVII (FVII) being the primary initiator of coagulation.

TABLE 3 Coagulation Factors Factor Common Name Pathway Characteristic IFibrinogen Both — II Prothrombin Both Contains N-terminal Gla domain IIITissue Factor Extrinsic — IV Calcium Both — V Proaccelerin, labilefactor, Both Protein cofactor Accelerator globulin VI (Va) Accelerin —(Redundant to factor V) VII Proconvertin, serum prothrombin ExtrinsicEndopeptidase with conversion accelerator (SPCA) Gla domaincothromboplastin VIII Antihemophiliac factor A, Intrinsic Proteincofactor antihemophiliac globulin (AHG) IX Christmas factor,antihemophiliac Intrinsic Endopeptidase with factor B, plasmathromboplastin Gla domain component (PTC) X Stuart-prower factor BothEndopeptidase with Gla domain XI Plasma thromboplastin IntrinsicEndopeptidase antecedent (PTA) XII Hageman factor IntrinsicEndopeptidase XIII Protransglutamidase, fibrin Both Transpeptidasestabilizing factor (FSF), fibrinoligase *Table adapted from M. W. King(2006) at med.unibs.it/~marchesi/blood.html

The generation of thrombin has historically been divided into threepathways, the intrinsic (suggesting that all components of the pathwayare intrinsic to plasma) and extrinsic (suggesting that one or morecomponents of the pathway are extrinsic to plasma) pathways that providealternative routes for the generation of activated factor X (FXa), andthe final common pathway which results in thrombin formation (FIG. 1).These pathways participate together in an interconnected andinterdependent process to effect coagulation. A cell-based model ofcoagulation was developed that describes these pathways (FIG. 2)(Hoffman et al. (2001) Thromb Haemost 85:958-965). In this model, the“extrinsic” and “intrinsic” pathways are effected on different cellsurfaces, the tissue factor (TF)-bearing cell and the platelet,respectively. The process of coagulation is separated into distinctphases, initiation, amplification and propagation, during which theextrinsic and intrinsic pathways function at various stages to producethe large burst of thrombin required to convert sufficient quantities offibrinogen to fibrin for clot formation.

a. Initiation

FVII is considered to be the coagulation factor responsible forinitiating the coagulation cascade, which initiation is dependent on itsinteraction with TF. TF is a transmembrane glycoprotein expressed by avariety of cells such as smooth muscle cells, fibroblasts, monocytes,lymphocytes, granulocytes, platelets and endothelial cells. Myeloidcells and endothelial cells only express TF when they are stimulated,such as by proinflammatory cytokines. Smooth muscle cells andfibroblasts, however, express TF constitutively. Accordingly, once thesecells come in contact with the bloodstream following tissue injury, thecoagulation cascade is rapidly initiated by the binding of TF withfactor VII or FVIIa in the plasma.

As discussed below, the majority of FVII in the blood is in the zymogenform with a small amount, approximately 1%, present as FVIIa. In theabsence of TF binding, however, even FVIIa has zymogen-likecharacteristics and does not display significant activity until it iscomplexed with TF. Thus, plasma FVII requires activation by proteolyticcleavage, and additional conformational change through interaction withTF, for full activity. A range of proteases, including factors IXa, Xa,XIIa, and thrombin, have been shown to be capable of FVII cleavage invitro, a process which is accelerated in the presence of TF. FVIIaitself also can activate FVII in the presence of TF, a process termedautoactivation. The small amounts of FVIIa in the blood are likely dueto activation by FXa and/or FIXa (Wildgoose et al. (1992) Blood80:25-28, and Butenas et al. (1996) Biochemistry 35:1904-1910). TF/FVIIacomplexes can thus be formed by the direct binding of FVIIa to TF, or bythe binding of FVII to TF and then the subsequent activation of FVII toFVIIa by a plasma protease, such as FXa, FIXa, FXIa, or FVIIa itself.The TF/FVIIa complex remains anchored to the TF-bearing cell where itactivates small amounts FX into FXa in what is known as the “extrinsicpathway” of coagulation.

The TF/FVIIa complex also cleaves small amounts of FIX into FIXa. FXaassociates with its cofactor FVa to also form a complex on theTF-bearing cell that can then covert prothrombin to thrombin. The smallamount of thrombin produced is, however, inadequate to support therequired fibrin formation for complete clotting. Additionally, anyactive FXa and FIXa are inhibited in the circulation by antithrombin III(AT-III) and other serpins, which are discussed in more detail below.This would normally prevent clot formation in the circulation. In thepresence of injury, however, damage to the vasculature results inplatelet aggregation and activation at this site of thrombin formation,thereby allowing for amplification of the coagulation signal.

b. Amplification

Amplification takes place when thrombin binds to and activates theplatelets. The activated platelets release FV from their alpha granules,which is activated by thrombin to FVa. Thrombin also releases andactivates FVIII from the FVIII/vWF complex on the platelet membrane, andcleaves FXI into FXIa. These reactions generate activated platelets thathave FVa, FVIIIa and FIXa on their surface, which set the stage for alarge burst of thrombin generation during the propagation stage.

c. Propagation

Propagation of coagulation occurs on the surface of large numbers ofplatelets at the site of injury. As described above, the activatedplatelets have FXIa, FVIIIa and FVa on their surface. It is here thatthe extrinsic pathway is effected. FXIa activates FIX to FIXa, which canthen bind with FVIIIa. This process, in addition to the small amounts ofFIXa that is generated by cleavage of FIX by the TF/FVIIa complex on theTF-bearing cell, generates large numbers of FXIa/FVIIIa complexes whichin turn can activate significant amounts of FX to FXa. The FXa moleculesbind to FVa to generate the prothrombinase complexes that activateprothrombin to thrombin. Thrombin acts in a positive feedback loop toactivate even more platelets and again initiates the processes describedfor the amplification phase.

Very shortly, there are sufficient numbers of activated platelets withthe appropriate complexes to generate the burst of thrombin that islarge enough to generate sufficient amounts of fibrin from fibrinogen toform a hemostatic fibrin clot. Fibrinogen is a dimer soluble in plasmawhich, when cleaved by thrombin, releases fibrinopeptide A andfibrinopeptide B. Fibrinopeptide B is then cleaved by thrombin, and thefibrin monomers formed by this second proteolytic cleavage spontaneouslyforms an insoluble gel. The polymerized fibrin is held together bynoncovalent and electrostatic forces and is stabilized by thetransamidating enzyme factor XIIIa (FXIIIa), produced by the cleavage ofFXIII by thrombin. Thrombin also activates TAFI, which inhibitsfibrinolysis by reducing plasmin generation at the clot surface.Additionally, thrombin itself is incorporated into the structure of theclot for further stabilization. These insoluble fibrin aggregates(clots), together with aggregated platelets (thrombi), block the damagedblood vessel and prevent further bleeding.

3. Regulation of Coagulation

During coagulation, the cascade is regulated by constitutive andstimulated processes to inhibit further clot formation. There areseveral reasons for such regulatory mechanisms. First, regulation isrequired to limit ischemia of tissues by fibrin clot formation. Second,regulation prevents widespread thrombosis by localizing the clotformation only to the site of tissue injury.

Regulation is achieved by the cations of several inhibitory molecules.For example, antithrombin III (AT-III) and tissue factor pathwayinhibitor (TFPI) work constitutively to inhibit factors in thecoagulation cascade. AT-III inhibits thrombin, FIXa, and FXa, whereasTFPI inhibits FXa and FVIIa/TF complex. An additional factor, Protein C,which is stimulated via platelet activation, regulates coagulation byproteolytic cleavage and inactivation of FVa and FVIIIa. Protein Senhances the activity of Protein C. Further, another factor whichcontributes to coagulation inhibition is the integral membrane proteinthrombomodulin, which is produced by vascular endothelial cells andserves as a receptor for thrombin. Binding of thrombin to thrombomodulininhibits thrombin procoagulant activities and also contributes toprotein C activation.

Fibrinolysis, the breakdown of the fibrin clot, also provides amechanism for regulating coagulation. The crosslinked fibrin multimersin a clot are broken down to soluble polypeptides by plasmin, a serineprotease. Plasmin can be generated from its inactive precursorplasminogen and recruited to the site of a fibrin clot in two ways: byinteraction with tissue plasminogen activator (tPA) at the surface of afibrin clot, and by interaction with urokinase plasminogen activator(uPA) at a cell surface. The first mechanism appears to be the major oneresponsible for the dissolution of clots within blood vessels. Thesecond, although capable of mediating clot dissolution, can play a majorrole in tissue remodeling, cell migration, and inflammation.

Clot dissolution also is regulated in two ways. First, efficient plasminactivation and fibrinolysis occur only in complexes formed at the clotsurface or on a cell membrane, while proteins free in the blood areinefficient catalysts and are rapidly inactivated. Second, plasminogenactivators and plasmin are inactivated by molecules such as plasminogenactivator inhibitor type 1 (PAI-1) and PAI-2 which act on theplasminogen activators, and α2-antiplasmin and α2-macroglobulin thatinactivate plasmin. Under normal circumstances, the timely balancebetween coagulation and fibrinolysis results in the efficient formationand clearing of clots following vascular injury, while simultaneouslypreventing unwanted thrombotic or bleeding episodes.

A summary of exemplary coagulation factors, cofactors and regulatoryproteins, and their activities, are set forth in Table 4 below.

TABLE 4 Coagulation Factor Zymogens and Cofactors Name of FactorActivity Zymogens of Serine Proteases Factor XII Binds exposed collagenat site of vessel wall injury, activated by high-MW kininogen andkallikrein Factor XI Activated by factor XIIa Factor IX Activated byfactor XIa + Ca²⁺ Factor VII Activated by thrombin, factor X, factor IXaor factor XIIa + Ca²⁺, or autoactivation Factor X Activated on plateletsurface by tenase complex (FIXa/FVIIIa); Also activated by factor VIIa +tissue factor + Ca²⁺, or factor VIIa + Ca²⁺ Factor II Activated onplatelet surface by prothrombinase complex (FXa/FVa) Cofactors FactorVIII Activated by thrombin; factor VIIIa acts as cofactor for factor IXain activation of factor X Factor V Activated by thrombin; factor Va actsas cofactor for factor Xa in activation of prothrombin Factor III(Tissue factor) Acts as cofactor for factor VIIa Fibrinogen Factor I(Fibrinogen) Cleaved by thrombin to form fibrin Transglutaminase FactorXIII Activated by thrombin + Ca²⁺; promotes covalent cross-linking offibrin Regulatory and other proteins von Willebrand factor (vWF) Acts asbridge between GPIb-V-IX complex and collagen Protein C Activated bythrombin bound to thrombomodulin; Ca degrades factors VIIIa and VaProtein S Acts as cofactor of protein C Thrombomodulin Endothelial cellsurface protein; binds thrombin, which activates protein C AntithrombinIII Coagulation inhibitor, primarily of thrombin and factor Xa, but alsofactors IXa, XIa, and XIIa, and factor VIIa complexed with TF TissueFactor Pathway Binds FXa and then forms a quaternary Inhibitor (TFPI)structure with TF/FVIIa to inhibit TF/FVIIa activity *Table adapted fromM. W. King (2006) at med.unibs.it/~marchesi/blood.html

C. Factor VII (FVII)

Factor VII is a vitamin K-dependent serine protease glycoprotein that issynthesized in animals, including mammals, as a single-chain zymogen inthe liver and secreted into the blood stream. As described above, FVIIis the coagulation protease responsible for initiating the cascade ofproteolytic events that lead to thrombin generation and fibrindeposition. It is part of the extrinsic pathway, although the downstreameffects of its activity also impact greatly on the intrinsic pathway.This integral role in clot formation has attracted significant interestin FVII as a target for clinical anti-coagulant and haemostatictherapies. For example, recombinant activated FVII (rFVIIa) has beendeveloped as a haemostatic agent for use in hemophilic subjects, andsubjects with other bleeding conditions. Provided herein are modifiedFVII polypeptides that are designed to have increased coagulationactivity upon activation, and that can serve as improved therapeutics totreat diseases and conditions amenable to factor VII therapy.

1. FVII Structure and Organization

The human FVII gene (F7) is located on chromosome 13 at 13q34 and is12.8 kb long with 9 exons. The FVII gene shares significantorganizational similarity with genes coding for other vitamin-Kdependent proteins, such as prothrombin, factor IX, factor X and proteinC. The mRNA for FVII undergoes alternative splicing to produce twotranscripts: variant 1 (Genbank Accession No. NM_(—)000131, set forth inSEQ ID NO:81) and variant 2 (Genbank Accession No. NM_(—)019616, setforth in SEQ ID NO:82). Transcript variant 2, which is the more abundantform in the liver, does not include exon 1b and thus encodes a shorterprecursor polypeptide of 444 amino acids (FVII isoform b precursor; SEQID NO:2), compared with the 466 amino acid precursor polypeptide encodedby transcript variant 1 (FVII isoform a precursor; SEQ ID NO:1). Theamino acids that are not present in the FVII isoform b precursorpolypeptide correspond to amino acid positions 22 to 43 of the FVIIisoform a precursor. These amino acids are part of the propeptidesequence, resulting in truncated FVII isoform b propeptide. Theprecursor polypeptides are made up of the following segments anddomains: a hydrophobic signal peptide (aa 1-20 of SEQ ID NO:1 and 2), apropeptide (aa 21-60 of SEQ ID NO:1, and aa 21-38 of SEQ ID NO:2), a Gladomain (aa 39-83 of SEQ ID NO:2, and aa 61-105 of SEQ ID NO:1), a type Bepidermal growth factor domain (EGF-like 1, aa 84-120 of SEQ ID NO:2,and aa 106-142 of SEQ ID NO:1), a type A epidermal growth factor domain(EGF-like 2, aa 125-166 of SEQ ID NO:2; and aa 147-188 of SEQ ID NO:1),and a serine protease domain (aa 191-430 of SEQ ID NO:2, and aa 213-452of SEQ ID NO:1).

The 406 amino acid mature form of the FVII polypeptide (SEQ ID NO:3)lacks the signal peptide and propeptide sequences, and is identical inlength and sequence regardless of the isoform precursor from which itoriginated. In the mature form of the FVII polypeptide the correspondingamino acid positions for the above mentioned domains are as follows: Gladomain (aa 1-45 of SEQ ID NO:3), EGF-like 1 (aa 46-82 of SEQ ID NO:3),EGF-like 2 (aa 87-128 of SEQ ID NO:3), and serine protease domain (aa153-392 of SEQ ID NO:3).

The Gla domain of FVII is a membrane binding motif which, in thepresence of calcium ions, interacts with phospholipid membranes thatinclude phosphatidylserine. The Gla domain also plays a role in bindingto the FVIIa cofactor, tissue factor (TF). Complexed with TF, the Gladomain of FVIIa is loaded with seven Ca²⁺ ions, projects threehydrophobic side chains in the direction of the cell membrane forinteraction with phospholipids on the cell surface, and has significantcontact with the C-terminal domain of TF. The Gla domain is conservedamong vitamin K-dependent proteins, such as prothrombin, coagulationfactors VII, IX and X, proteins C, S, and Z. These proteins requirevitamin K for the posttranslational synthesis of γ-carboxyglutamic acid,an amino acid clustered in the N-terminal Gla domain of these proteins.All glutamic residues present in the domain are potential carboxylationsites and many of them are therefore modified by carboxylation.

In addition to the Gla domain, the mature FVII protein also contains twoEGF-like domains. The first EGF-like domain (EGF-like 1 or EGF1) is acalcium-binding EGF domain, in which six conserved core cysteines formthree disulfide bridges. The EGF1 domain of FVII binds just one Ca²⁺ion, but with significantly higher affinity than that observed with theGla domain (Banner et al. (1996) Nature 380:41-46). This bound Ca²⁺ ionpromotes the strong interaction between the EGF1 domain of FVII and TF(Osterlund et al. (2000) Eur J Biochem 267:6204-6211.) The secondEGF-like domain (EGF-like 2 or EGF2) is not a calcium-binding domain,but also forms 3 disulphide bridges. Like the other domains in FVII, theEGF2 domain interacts with TF. It also is disulphide-bonded togetherwith the protease domain, with which it shares a large contactinterface.

Finally, the serine protease domain of FVII is the domain responsiblefor the proteolytic activity of FVIIa. The sequence of amino acids ofFVII in its catalytic domain displays high sequence identity andtertiary structure similarity with other serine proteases such astrypsin and chymotrypsin (Jin et al. (2001) J Mol Biol, 307: 1503-1517).For example, these serine proteases share a common catalytic triad H57,D102, S195, based on chymotrypsin numbering. Unlike other serineproteases, however, cleavage of FVIIa is not sufficient to complete theconversion of the zymogen to a fully active enzyme. Instead, asdiscussed below, FVIIa is allosterically activated in its catalyticfunction by binding to the cell-surface receptor TF, which induces aconformational change in the FVIIa protease domain switching it from azymogen-like inactive state to a catalytically active enzyme. A helixloop region between the cofactor binding site and the active site (i.e.amino acid residue positions 305-321, corresponding to residues 163-170ibased on chymotrypsin numbering) of FVIIa is important for the allosteryand zymogenicity of FVIIa (Persson et al. (2004) Biochem J., 379:497-503). This region is composed of a short a helix (amino acid residuepositions 307 to 312) followed by a loop. The N-terminal portion of thehelix forms part of the interface between the protease domain and TF,and contains a number of residues that are important for proteolyticfunction and optimal binding to TF. A comparison of the crystalstructure of FVIIa alone and FVIIa complexed with TF indicates that theα helix undergoes significant conformational change when FVIIa binds TF.The α helix of FVIIa alone appears distorted, shortened and orienteddifferently. This affects adjacent loop structures, moving them awayfrom the active site. In contrast, the α helix of FVIIa when complexedwith TF is stabilized, and the neighboring loops are positioned closerto the active site. This stabilization is effected through mechanismsthat involve at least the methionine at amino acid position 306 (aminoacid residue Met164 by chymotrypsin numbering) of FVII (Pike et al.(1999) PNAS 8925-8930).

2. Post-Translational Modifications

The FVII precursor polypeptide (either isoform of the Factor VII gene)is targeted to the cellular secretory pathway by the hydrophobic signalpeptide, which inserts into the endoplasmic reticulum (ER) to initiatetranslocation across the membrane. While the protein is translocatedthrough the ER membrane, the 20 amino acid signal peptide is cleaved offby a signal peptidase within the ER lumen, after which the polypeptideundergoes further post-translational modifications, including N- andO-glycosylation, vitamin K-dependent carboxylation of N-terminalglutamic acids to γ-carboxyglutamic acids, and hydroxylation of asparticacid to β-hydroxyaspartic acid.

The propeptide provides a binding site for a vitamin K-dependentcarboxylase which recognizes a 10-residue amphipathic α-helix in theFVII propeptide. After binding, the carboxylase γ-carboxylates 10glutamic acid residues within the Gla domain of the FVII polypeptide,producing γ-carboxyglutamyl residues at positions E66, E67, E74, E76,E79, E80, E85, E86, E89 and E95 relative to the FVII precursor aminoacid sequence set forth in SEQ ID NO:2. These positions correspond topositions E6, E7, E14, E19, E20, E25, E26, E29 and E35 of the matureFVII polypeptide set forth in SEQ ID NO:3. For optimal activity, theFVII molecule requires calcium, which binds the polypeptide andfacilitates the conformational changes needed for binding of FVIIa withTF and lipids. The γ-carboxylated Gla domain binds seven Ca²⁺ ions withvariable affinity, which induces the conformational change that enablesthe Gla domain to interact with the C-terminal domain of TF, and alsophosphatidylserines or other negatively charged phospholipids on theplatelet membrane.

N-linked glycosylation is carried out by transfer of Glc₃Man₉ (GlcNAc)to two asparagine residues in the FVII polypeptide, at positions thatcorrespond to amino acid residues 145 and 322 of the mature protein (SEQID NO:3). O-linked glycosylation occurs at amino acid residues 52 and 60of the mature polypeptide, and hydroxylation to a β-hydroxyaspartic acidoccurs at the aspartic acid residue at position 63. These O-glycosylatedserine residues and the β-hydroxylated aspartic acid residue are in theEGF-1 domain of FVII. These modifications are effected in the ER andGolgi complex before final processing of the polypeptide to its matureform.

3. FVII Processing

The modified pro-FVII polypeptide is transported through the Golgi lumento the trans-Golgi compartment where the propeptide is cleaved by apropeptidase just prior to secretion of the protein from the cell.PACE/furin (where PACE is an acronym for Paired basic Amino acidCleaving Enzyme) is an endopeptidase localized to the Golgi membranethat cleaves many proteins on the carboxyterminal side of the sequencemotif Arg-[any residue]-(Lys or Arg)-Arg. This propeptidase cleavesvitamin K-dependent glycoproteins such as the pro-factor IX and pro-vWFpolypeptides (Himmelspach et al. (2000) Thromb Research 97; 51-67),releasing the propeptide from the mature protein. Inclusion of anappropriate PACE/furin recognition site into recombinant Factor VIIprecursors facilitates correct processing and secretion of therecombinant polypeptide (Margaritas et al. (2004) Clin Invest 113(7):1025-1031). PACE/furin, or another subtilising-like propeptidase enzyme,is likely responsible for the proteolytic processing of pro-FVII toFVII. It can recognize and bind to the -Arg-Arg-Arg-Arg- consensus motifat amino acid positions 35-38 of the sequences set forth in SEQ ID NO:1,and positions 57-60 of the sequence set forth in SEQ ID NO:2, cleavingthe propeptide and releasing the mature protein for secretion.

4. FVII Activation

The vast majority of FVII in the blood is in the form of an unactivatedsingle-chain zymogen, although a small amount is present in a two-chainactivated form. Activation of FVII occurs upon proteolytic cleavage ofthe Arg¹⁵²-Ile¹⁵³ bond (positions relative to the mature FVIIpolypeptide, set forth in SEQ ID NO:3), giving rise to a two-chainpolypeptide containing a 152 amino acid light chain (approximately 20kDa) linked by a disulphide bridge to a 254 amino acid heavy chain(approximately 30 kDa). The light chain of FVIIa contains the Gla domainand EGF-like domains, while the heavy chain contains the catalytic orserine-protease portion of the molecule. Conversion of the single chainFVII into the two-chain FVIIa is mediated by cleavage by FIXa, FXa,FXIIa, thrombin, or in an autocatalytic manner by endogenous FVIIa(Butenas et al. (1996) Biochem 35:1904-1910; Nakagaki et al. (1991)Biochem 30:10819-10824). The trace amount of FVIIa that does occur incirculation likely arises from the action of FXa and FIXa.

As discussed above, cleavage of FVII from its zymogen form to FVIIa isnot sufficient for full activity. FVIIa requires association with TF forfull activity (Higashi et al. (1996) J Biol Chem 271:26569-26574).Because of this requirement, FVIIa alone has been ascribed zymogen-likefeatures, displaying zymogen folding and shape, and exhibitingrelatively low activity. This zymogen-like characteristic of FVIIa inthe absence of its association with TF makes it relatively resistant toantithrombin III (AT-III) and other serpins, which generally actprimarily on the active forms of serine proteases rather than thezymogen form. In addition, TFPI, the principal inhibitor of TF/FVIIaactivity, also does not bind efficiently to the “inactive” uncomplexedform of FVIIa.

Upon complexation with TF, FVIIa undergoes a conformational change thatpermits full activity of the molecule. All of the FVII domains areinvolved in the interaction with TF, but the conformational changes thatoccur are localized to the protease domain of FVIIa. For example, theconformational changes that occur in upon allosteric interaction ofFVIIa and TF include the creation of an extended macromolecularsubstrate binding exosite. This extended binding site greatly enhancesthe FVII-mediated proteolytic activation of factor X.

The activity of FVIIa is further increased (i.e. a thousand-fold) whenthe interaction of FVIIa is with cell surface-expressed TF. This isbecause phospholipid membranes containing negatively-chargedphospholipids, such as phosphatidylserine, are a site of interaction ofother vitamin-K dependent coagulation factors such as FIX and FX, whichbind via their Gla domains. Thus, the local concentration of thesevitamin K-dependent proteins is high at the cell surface, promotingtheir interaction with the TF/FVIIa complex.

5. FVII Function

Although FVIIa exhibits increased activity following allostericactivation by TF, there is evidence that mechanisms exist in which FVIIaalone can initiate coagulation. Hence, FVII can function in aTF-dependent and a TF-independent manner. This latter pathway can play amuch smaller role in normal hemostasis, although its significance couldincrease when it is considered in the context of bleeding disorders, andthe treatment thereof.

a. Tissue Factor-Dependent FVIIa Activity

Circulating FVII binds cell-surface TF and is activated by FIXa, FXa,thrombin, or in an autocatalytic manner by endogenous FVIIa as describedabove. Alternatively, the very small amount of circulating FVIIa candirectly bind TF. The TF/FVIIa complex then binds a small fraction ofplasma FX and the FVIIa catalytic domain cleaves FX to produce FXa.Thrombin is thus formed via the extrinsic pathway on the surface of theTF-bearing cell, when FXa complexes with FVa and activates prothrombinto thrombin (FIG. 3). FIX also is activated by the TF/FVIIa complex,providing a link to the intrinsic pathway that operates on the surfaceof the activated platelet. The positive feedback systems in thecoagulation cascade described above provide the means by which largeamounts of thrombin are produced, which cleaves fibrinogen into fibrinto form a clot.

b. Tissue Factor-Independent FVIIa Activity

In addition to the TF-dependent mechanism for the activation of FX toFXa, there is evidence that FVIIa also can activate FX in the absence ofTF. Activated platelets translocate phosphatidylserines and othernegatively charged phospholipids to the outer, plasma-oriented surface.(Hemker et al. (1983) Blood Cells 9:303-317). These provide alternative“receptors” through which FVIIa can bind, albeit with a relatively lowaffinity that is 1000-fold less than the binding affinity of FVIIa to TF(Monroe et al. (1997) Br J Haematol 99:542-7). This interaction ismediated through residues in the Gla domain (Harvey et al. (2003)278:8363-8369). FVIIa can then convert FX to FXa and FIX to FIXa on theactivated platelet surface (Hoffman et al. (1998) Blood CoagulFibrinolysis 9:S61-S65). The FXa remains associated with the plateletsurface, where it can bind to FVa and generate sufficient thrombin fromprothrombin, while the newly formed FIXa assembles with FVIIIa tocatalyze the activation of more FX to FXa (FIG. 3). Hemostasis in theabsence of TF can then achieved by the positive feedback and propagationmechanisms described above. It is notable, however, that while FVIIIacan contribute to the coagulation process on the activated platelet, itspresence is not required for thrombin generation in the TF-independentmechanism (FIG. 3). Thus, in the absence of FVIII, such as in hemophiliapatients, there is evidence that FVIIa can initiate and/or amplifythrombin generation through this secondary mechanism, and effect clotformation.

6. FVII as a Biopharmaceutical

FVII functions to initiate blood coagulation. Recombinant FVIIa(NovoSeven®; rFVIIa) is approved for treatment of bleeding episodes orprevention of bleeding in surgical or invasive procedures in patientshaving hemophilia A or B with inhibitors to Factor VIII or Factor IX,and in patients with congenital Factor VII deficiency. Novoseven® is agenetically engineered preparation of factor VIIa that is produced in amammalian expression system using baby hamster kidney (BHK) cells. Theagent is nearly identical to plasma-derived factor VIIa in its structureand function (Ratko et al. (2004), P & T, 29: 712-720).

Administration of recombinant FVIIa (rFVIIa) has been shown to promoteblood clotting in patients suffering from hemophilia, and treatment withdoses of FVIIa have been found to be safe and well-tolerated in humansubjects. Typically, the use of rFVIIa has been in patients who havedeveloped inhibitors (i.e. alloantibodies) to Factor VIII or Factor IX.The use of rFVIIa as a coagulant has been extended to treatment of otherbleeding disorders, for example Glanzmann's thrombasthenia; other eventsassociated with extensive bleeding, such as a result of trauma orsurgery including, but not limited to, liver transplants, prostatesurgery and hemorrhaging trauma; neonatal coagulophathies, severehepatic disease; bone marrow transplantation, thrombocytopenias andplatelet function disorders; urgent reversal of oral anticoagulation;congenital deficiencies of factors V, VII, X, and XI; and von Willebranddisease with inhibitors to von Willebrand factor.

A high-dose of rFVII is required to achieve a therapeutic effect. Thedose and dosing regime required for rFVII administration variesdepending on the clinical indication. For example, the typical dosage ofrFVII for hemorrhagic episodes in patients with hemophilia A orhemophilia B having alloantibodies is 90 μg/kg administered byintravenous (IV) injection. Since rFVII has a half-life of 2 hours,repeat dosing is required. Additional dosing can be given every twohours until hemostasis is achieved. The dose range can be altereddepending on the severity of the condition. For example, doses rangingfrom 35-120 μg/kg have been efficacious. Also, the dose and dosingregime can vary with other indications. For example, hemophilia A orhemophilia B patients undergoing surgery can be administered with aninitial dose of 90 μg/kg immediately before surgery, with repeat dosinggiven every two hours during and following surgery. Depending on theseverity of the surgery and bleeding episode, the bolus IV infusion cancontinue every two to six hours until healing is achieved. In congenitalFVII deficient patients, rFVII is typically administered to preventbleeding in surgery or other invasive procedures at 15-30 μg/kg every4-6 hours until hemostasis is achieved.

The mechanism of action of rFVIIa to initiate hemostasis explains thehigh-dose requirement. Hemophilia patients have a normal initiationphase of coagulation, where the TF/FVIIa complex activates FX to FXa andleads to thrombin production at the site of the TF-bearing cell.Thereafter, however, the coagulation process breaks down as hemophiliapatients lack FVIII (hemophilia A) or FIX (hemophilia B), and aretherefore unable to form the FVIIIa/FIXa complexes on the surface of theactivated platelet, which normally serve to activate large amounts of FXto FXa in the amplification and propagation phases described previously.Due to the presence of inhibitors, such as TFPI and AT-III, the FXa thatis produced on the TF-bearing cell following cleavage by TF/FVIIa isunable to easily diffuse between cell surfaces. As a result, large-scalethrombin generation on the surface of the activated platelet does notoccur, and a clot is not formed.

There is evidence that the hemostatic effect of high doses of rFVIIa canbe achieved using TF-dependent and/or TF-independent generation of FXaby rFVIIa on the activated platelets (FIG. 3). TF-dependent thrombingeneration can be maximized very quickly with the saturation of TFmolecules with endogenous FVIIa and rFVIIa. In some instances, the highdose rFVIIa can bind activated platelets and convert FX to FXa. Thesurface-associated FXa activates FVa to generate sufficient thrombin forhemostasis. Since rFVII binds to the platelet surface with low affinity,a higher dose of rFVII can be required for thrombin generation. Theactivation of FXa on activated platelets ensures that rFVIIa-mediatedhemostasis is localized to the site of injury.

A means to achieve reduced dosage of rFVII can improve its utility andefficiency as a drug. Provided herein are modified FVII polypeptides.Among these are modified FVII polypeptides that exhibit increasedresistance to AT-III and increased catalytic activity in the presenceand/or absence of TF. The modified FVII polypeptides provided hereinalso can exhibit increased resistance to TFPI, increased resistance tothe inhibitory effects of Zn²⁺, improved pharmacokinetic properties,such as increased serum half-life, increased binding and/or affinity foractivated platelets, increased binding and/or affinity for serumalbumin, and/or increased binding and/or affinity for platelet integrinα_(IIb)β₃. These modified FVII polypeptides can exhibit increasedcoagulant activity. FVII polypeptides provided herein can be used intreatments to initiate hemostasis in a TF-dependent and/or aTF-independent mechanism such that FXa is produced and thrombingenerated.

D. Modified FVII Polypeptides

Provided herein are modified FVII polypeptides. The FVII polypeptidesexhibit alterations in one or more activities or properties compared toFVII polypeptide that is not so-modified. The activities or propertiesthat can be altered as a result of modification include, but are notlimited to, coagulation or coagulant activity; pro-coagulant activity;proteolytic or catalytic activity such as to effect factor X (FX)activation or Factor IX (FIX) activation; antigenicity (ability to bindto or compete with a polypeptide for binding to an anti-FVII antibody);ability to bind tissue factor, factor X or factor IX; ability to bind tophospholipids; half-life; three-dimensional structure; pI; and/orconformation. Typically, the modified FVII polypeptides exhibitprocoagulant activity. Provided herein are modified FVII polypeptidesthat exhibit increased coagulant activity upon activation from theirsingle-chain zymogen form. Such modified FVII polypeptides can be usedin the treatment of bleeding disorders or events, such as hemophilias orinjury, where FVII polypeptides can function to promote bloodcoagulation. Included among such modified FVII polypeptides are thosethat have increased resistance to inhibitors such as antithrombin III(AT-III) and tissue factor pathway inhibitor (TFPI), those that haveincreased resistance to the inhibitory effects of Zn²⁺, those that haveincreased catalytic activity in the presence and/or absence of TF, thosethat have improved pharmacokinetic properties, such as increasedhalf-life, those that have increased binding and/or affinity for theplatelet surface, those that have increased binding and/or affinity forserum albumin, and those that have increased binding and/or affinity forplatelet integrin α_(IIb)β₃. In particular, such modified FVIIpolypeptides can be used in diseases or conditions to provide coagulantactivity while at the same time bypassing the requirements for FVIIIaand FIXa. In one example, modified FVII polypeptides provided herein canbe used in hemophiliac patients having autoantibodies to FVIIIa andFIXa. Hence, the modified FVII polypeptides provided herein offeradvantages including a decrease in the amount of administered FVII thatis required to maintain a sufficient concentration of active FVII in theserum for hemostasis. This can lead to, for example, lower doses and/ordosage frequency necessary to achieve comparable biological effects,higher comfort and acceptance by subjects, and attenuation of secondaryeffects.

Modifications in a FVII polypeptide can be made to any form of a FVIIpolypeptide, including allelic and species variants, splice variants,variants known in the art, or hybrid or chimeric FVII molecules. Forexample, the modifications provided herein can be made in a precursorFVII polypeptide set forth in SEQ ID NOS:1 or 2, a mature FVIIpolypeptide set forth in SEQ ID NO:3, or any species, allelic ormodified variants and active fragments thereof, that has 40%, 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to any of the FVII polypeptides set forth in SEQ ID NOS:1-3.Allelic variants of FVII include, but are not limited to, any of thoseprecursor polypeptides having a sequence of amino acids set forth in anyof SEQ ID NOS:18-74. Exemplary species variants for modification hereininclude, but are not limited to, human and non-human polypeptidesincluding FVII polypeptides from cow, mouse, pygmy chimpanzee,chimpanzee, rabbit, rat, rhesus macaque, pig, dog, zebra fish,pufferfish, chicken, orangutan and gorilla FVII polypeptides, whosesequences are set forth in SEQ ID NOS: 4-17 respectively. Modificationsin a FVII polypeptide can be made to a FVII polypeptide that alsocontains other modifications, such as those described in the art,including modifications of the primary sequence and modifications not inthe primary sequence of the polypeptide.

Modification of FVII polypeptides also include modification ofpolypeptides that are hybrids of different FVII polypeptides and alsosynthetic FVII polypeptides prepared recombinantly or synthesized orconstructed by other methods known in the art based upon the sequence ofknown polypeptides. For example, based on alignment of FVII with othercoagulation factor family members, such as factor IX (FIX) or factor X(FX), homologous domains among the family members are readilyidentified. Chimeric variants of FVII polypeptides can be constructedwhere one or more amino acids or entire domains are replaced in the FVIIamino acid sequence using the amino acid sequence of the correspondingfamily member. Additionally, chimeric FVII polypeptides include thosewhere one or more amino acids or entire domains are replaced in thehuman FVII amino acid sequence using the amino acid sequence of adifferent species (see, e.g., Williamson et al. (2005) J Thromb Haemost3:1250-6). Such chimeric proteins can be used as the starting,unmodified FVII polypeptide herein.

Modifications provided herein of a starting, unmodified referencepolypeptide include amino acid replacements or substitution, additionsor deletions of amino acids, or any combination thereof. For example,modified FVII polypeptides include those with 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more modifiedpositions. Also provided herein are modified FVII polypeptides with twoor more modifications compared to a starting reference FVII polypeptide.Modified FVII polypeptides include those with 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more modifiedpositions. Any modification provided herein can be combined with anyother modification known to one of skill in the art so long as theresulting modified FVII polypeptide exhibits increased coagulationactivity when it is in its two-chain form. Typically, the modified FVIIpolypeptides exhibit increased coagulant activity. The activities orproperties that can be altered as a result of modification include, butare not limited to, coagulation or coagulant activity; pro-coagulantactivity; proteolytic or catalytic activity such as to effect factor X(FX) activation or Factor IX (FIX) activation; antigenicity (ability tobind to or compete with a polypeptide for binding to an anti-FVIIantibody); ability to bind tissue factor, tissue factor inhibitoryfactor (TFPI), antithrombin III, factor X or factor IX; ability to bindto phospholipids, serum albumin or platelet integrin α_(IIb)β₃; serumhalf-life; three-dimensional structure; pI; and/or conformation.Included among the modified FVII polypeptides provided herein are thosethat have increased resistance to antithrombin III (AT-III), increasedcatalytic activity in the presence and/or absence of TF, increasedresistance to tissue factor pathway inhibitor (TFPI), increasedresistance to the inhibitory effects of Zn²⁺, improved pharmacokineticproperties, such as increased serum half-life, increased intrinsicactivity, altered glycosylation, increased affinity and/or binding forserum albumin, increased affinity and/or binding for platelet integrinα_(IIb)β₃, and/or increased affinity and/or binding for activatedplatelets.

In some examples, a modification can affect two or more properties oractivities of a FVII polypeptide. For example, a modification can resultin increased AT-III resistance and increased catalytic activity of themodified FVII polypeptide compared to an unmodified FVII polypeptide.Modified FVII polypeptides provided herein can be assayed for eachproperty and activity to identify the range of effects of amodification. Such assays are known in the art and described below.Modified FVII polypeptides provided herein also include FVIIpolypeptides that are additionally modified by the cellular machineryand include, for example, glycosylated, γ-carboxylated andβ-hydroxylated polypeptides.

The modifications provided herein to a FVII polypeptide are made toincrease AT-III resistance, increase TFPI resistance, increaseresistance to the inhibitory effects of Zn²⁺, improve pharmacokineticproperties, such as increase serum half-life, increase catalyticactivity in the presence and/or absence of TF, increase binding toactivated platelets, alter glycosylation, increase affinity and/orbinding to platelet integrin α_(IIb)β₃, increase affinity and/or bindingto serum albumin, and/or increase affinity and/or binding for activatedplatelets. For example, a FVII polypeptide can include modification(s)that increase one or both of catalytic activity and binding toplatelets. In other examples, any modification provided herein can becombined with any other modification known to one of skill in the art solong as the resulting modified FVII polypeptide exhibits increasedcoagulation activity when it is in its two-chain form. Typically, suchincreased coagulation activity is due to increased resistance to AT-III,increased catalytic activity, increased resistance to the inhibitoryeffects of Zn²⁺, improved pharmacokinetic properties, such as increasedserum half-life, increased resistance to TFPI, altered glycosylation,increased binding and/or affinity for phospholipids, increased bindingand/or affinity for serum albumin, and/or increased binding and/oraffinity for platelet integrin α_(IIb)β₃. In some examples,modifications that are introduced into a FVII polypeptide to alter aspecific activity or property also, or instead, can affect anotheractivity or property. Thus, the modifications provided herein can affectthe property or activity that they were designed to affect and one ormore other properties or activities. For example, modifications made toa FVII polypeptide to increase catalytic activity also can increaseAT-III resistance. In some examples, a single modification, such assingle amino acid substitution, alters 2, 3, 4 or more properties oractivities of a FVII polypeptide. Modified FVII polypeptides providedherein can be assayed for each property and activity to identify therange of effects of a modification. Such assays are known in the art anddescribed below. Modified FVII polypeptides provided herein also includeFVII polypeptides that are additionally modified by the cellularmachinery and include, for example, glycosylated, γ-carboxylated andβ-hydroxylated polypeptides.

The modifications provided herein can be made by standard recombinantDNA techniques such as are routine to one of skill in the art. Anymethod known in the art to effect mutation of any one or more aminoacids in a target protein can be employed. Methods include standardsite-directed mutagenesis (using e.g., a kit, such as kit such asQuikChange available from Stratagene) of encoding nucleic acidmolecules, or by solid phase polypeptide synthesis methods. In addition,modified chimeric proteins provided herein (i.e. Gla domain swap) can begenerated by routine recombinant DNA techniques. For example, chimericpolypeptides can be generated using restriction enzymes and cloningmethodologies for routine subcloning of the desired chimeric polypeptidecomponents.

Other modifications that are or are not in the primary sequence of thepolypeptide also can be included in a modified FVII polypeptide, orconjugate thereof, including, but not limited to, the addition of acarbohydrate moiety, the addition of a polyethylene glycol (PEG) moiety,the addition of an Fc domain, etc. For example, such additionalmodifications can be made to increase the stability or half-life of theprotein.

The resulting modified FVII polypeptides include those that aresingle-chain zymogen polypeptide or those that are two-chainzymogen-like polypeptides. For example, any modified polypeptideprovided herein that is a single-chain polypeptide can be autoactivatedor activated by other coagulation factors to generate a modified FVIIthat is a two-chain form (i.e. FVIIa). The activities of a modified FVIIpolypeptide are typically exhibited in its two-chain form.

The modified FVII polypeptides provided herein can exhibit increasedAT-III resistance, increased catalytic activity in the presence and/orabsence of TF, increased resistance to the inhibitory effects of Zn²⁺,increased TFPI resistance, improved pharmacokinetic properties, such asincreased serum half-life, altered glycosylation, increased bindingand/or affinity for phospholipids, increased binding and/or affinity forserum albumin, and/or increased binding and/or affinity for plateletintegrin α_(IIb)β₃. Typically, such properties and/or activities of themodified FVII polypeptides provided herein are made while retainingother FVII activities or properties, such as, but not limited to,binding to TF and/or binding and activation of FX. Hence, modified FVIIpolypeptides provided herein retain TF binding and/or FX binding andactivation as compared to a wild-type or starting form of the FVIIpolypeptide. Typically, such activity is substantially unchanged (lessthan 1%, 5% or 10% changed) compared to a wild-type or starting protein.In other examples, the activity of a modified FVII polypeptide isincreased or is decreased as compared to a wild-type or starting FVIIpolypeptide. Activity can be assessed in vitro or in vivo and can becompared to the unmodified FVII polypeptide, such as for example, themature, wild-type native FVII polypeptide (SEQ ID NO:3), the wild-typeprecursor FVII polypeptide (SEQ ID NO:1 or 2), or any other FVIIpolypeptide known to one of skill in the art that is used as thestarting material.

Hence, by virtue of the modifications provided herein, the modified FVIIpolypeptides can exhibit increased coagulant activity, increasedduration of coagulant activity, and/or an enhanced therapeutic index.This can be observed in a TF-dependent and/or TF-independent manner.Typically, the increased coagulant activity, increased duration ofcoagulant activity, and/or an enhanced therapeutic index of the modifiedFVII polypeptides provided herein can be observed in vitro or ex vivo inappropriate assays, or in vivo, such as upon administration to asubject, such as a human or non-human subject. The increased activity ofthe modified FVII polypeptides can be increased by at least or about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%,300%; 400%, 500%, or more compared to the activity of the starting orunmodified FVIIa polypeptide.

1. Increased Catalytic Activity

FVII contains a serine residue (position 195 in standardchymotrypsin(ogen) numbering) in its active center that acts as anucleophile during the cleavage reaction. The catalytic triad of serineproteases also includes two additional residues: H57 and D102(chymotrypsin numbering). The catalytic triad of human FVIIa correspondsto H193, D242 and S344 of the mature FVII polypeptide set forth in SEQID NO:3. These three key amino acids each play an essential role in thecatalytic activity of the proteases. Serine proteases hydrolyze peptidebonds via the formation of tetrahedral transition states and acyl-enzymeintermediates. The reaction pathway begins with non-covalent binding ofthe substrate into a groove on the surface of the protease (i.e., theactive site cleft) that contains H57 and S195 to form a“Michaelis-Menton complex”. Productive progress along the reactionpathway requires subsequent, nucleophilic attack of the P1 carbonylresidue of the substrate by the O-gamma of the active site serine (i.e.,serine 195) of the enzyme to form a tetrahedral transition state thatrapidly converts into an acyl-enzyme intermediate. A structure withinthe active site cleft that includes residues glycine 193 and serine 195(corresponding to G342 and S344 of the mature FVII polypeptide set forthin SEQ ID NO:3) and is known as the oxyanion hole promotes efficientcatalysis by stabilizing the transition state. Specifically, the mainchain amide hydrogens of these two residues form stabilizing hydrogenbonds with the oxyanion (i.e., the carbonyl oxygen of the P1 residue)that is created in the tetrahedral transition state. In addition to thisstabilization, binding of the substrate within the oxyanion holepositions the scissile bond properly for the productive acylation anddeacylation reactions that result in bond cleavage. The importance ofthe oxyanion hole in FVII activity is highlighted by the observationthat mutations at amino acid position 342 (corresponding to 193 bychymotrypsin numbering) can result in FVII deficiency (see e.g. Bernardiet al., (1994) Br. J. Haematol. 86:610-618 and Bernardi et al., (1996)Human Mut. 8:108-115).

a. Exemplary Modifications to Increase Catalytic Activity

Provided herein are modified FVII polypeptides that exhibit increasedcoagulant activity. Such FVII polypeptides can be generated by aminoacid substitution of one or more residues that can affect theconformation of the oxyanion hole. The introduction of different aminoacid residues at particular positions (e.g., position 143 bychymotrypsin numbering, or 286 by mature FVII numbering) can alter theconformation of the modified FVII polypeptide such that the oxyanionhole is more effective during catalysis. This can result in a modifiedFVII polypeptide with increased catalytic activity compared to anunmodified FVII polypeptide. Changes in catalytic activity due tomutations affecting the oxyanion hole can manifest as increasedcoagulant activity. Increases in catalytic and coagulant activity of themodified FVII polypeptides provided herein can be observed in thepresence and/or absence of tissue factor (i.e. can be TF-dependentand/or TF-independent). Thus, when evaluated in an appropriate in vitro,in vivo, or ex vivo assay such as following administration to a subjectas a pro-coagulant therapeutic, the modified FVII polypeptides candisplay increased coagulant activity compared with that of theunmodified FVII polypeptides.

The conformation of the oxyanion hole can be altered to induce a moreeffective conformation by modification of one or more amino acidresidues that are involved in the formation of, or are in proximity to,the oxyanion hole. As provided herein, exemplary of such amino acidresidues is Q286 (numbering corresponding a mature FVII polypeptide setforth in SEQ ID NO:3), which corresponds to Q143 by chymotrypsinnumbering. Q286 can be modified by, for example, amino acidsubstitution, deletion or insertion. When the modification is effectedby amino acid substitution, the glutamine residue at position 286 can bereplaced with any other amino acid residue.

Q286 is located adjacent to and in contact with residues that formregions of the active site and active site cleft of the FVIIpolypeptide. As such, it has been stated that modification at thisposition should result in reduced catalytic activity (see e.g., U.S.Pat. No. 6,806,063). This has been demonstrated in previous studies(see, e.g., International Pat. Pub. No. WO2007031559), where theglutamine residue was replaced with an alanine (Q286A). The resultingmodified FVIIa polypeptide exhibits a reduced ability to activate FactorX compared with the wild-type polypeptide. In other studies, the samemutation had essentially no effect on catalytic activity of the FVIIamutant for Factor X (Dickinson et al., (1996) Proc. Nat. Acad. Sci. USA.93:14379-14384) or a synthetic substrate (International Pat. Pub. No.WO2007031559).

As demonstrated herein (see Example 4 and below), however, modificationof the FVII polypeptide at position 286 (numbering corresponding amature FVII polypeptide set forth in SEQ ID NO:3; corresponding toposition 143 by chymotrypsin numbering), particularly with a basicresidue, such as arginine (Arg, R), results in a modified FVIIpolypeptide with increased catalytic and coagulant activity.

Thus, provided herein are modified FVII polypeptides that contain amodification, such as amino acid replacement with a basic amino acid, atthe amino acid position corresponding to amino acid position 286 of amature FVII polypeptide set forth in SEQ ID NO:3 (amino acid position143 by chymotrypsin numbering). The modifications provided herein atamino acid position 286 can be made in any FVII polypeptide, including aprecursor FVII polypeptide set forth in SEQ ID NOS:1 or 2, a mature FVIIpolypeptide set forth in SEQ ID NO:3, or any species, allelic ormodified variants and active fragments thereof, that has 40%, 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to any of the FVII polypeptides set forth in SEQ ID NOS:1-3.

Modification of a FVII polypeptide at amino acid position 286 by matureFVII numbering (corresponding to amino acid 143 by chymotrypsinnumbering) can alter the conformation of the oxyanion hole to aconformation that facilitates more effective catalysis of a substrate.Increased catalytic activity of such modified FVII polypeptides can beexhibited in the presence and/or absence of tissue factor, and can beassessed using in vitro assays such as those described in Examples 4 and7, below. In addition to exhibiting increased catalytic activity, FVIIpolypeptides that have been modified at amino acid position 286 bymature FVII numbering also can exhibit increased resistance to AT-III.This can be due to, for example, reduced binding of the modified FVIIpolypeptide to AT-III under specified conditions (e.g., followinginjection into a patient) or a reduced rate of inactivation by ATIII(i.e., a reduced second order rate constant for inhibition), which canmanifest as increased coagulant activity in the presence of AT-IIIcompared to an unmodified FVII polypeptide. Increased resistance toAT-III can be assessed using in vitro assays such as that described inExample 5.

Amino acid residue Q286 by mature FVII numbering (corresponding to Q143by chymotrypsin numbering) can be modified by amino acid deletion, orreplacement or substitution with any other amino acid. Alternatively, anamino acid can be inserted immediately before or after to alter theconformation in the vicinity of amino acid residue Q286. Further, a FVIIpolypeptide containing a modification of Q286 also can contain one ormore other modifications, including amino acid insertions, deletions,substitutions or replacements, and modifications not in the primarysequence of the polypeptide, such as the addition of a carbohydratemoiety, the addition of a polyethylene glycol (PEG) moiety, the additionof an Fc domain, etc., or any combination thereof. Thus, a FVIIpolypeptide containing a modification at amino acid position 286 bymature FVII numbering can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more modified positions.Such polypeptides retain at least one activity of an unmodified FVIIpolypeptide. Typically, the modified FVII polypeptide exhibits increasedcoagulant activity.

These changes in activities can manifest as increased coagulantactivity, increased duration of coagulant activity, increased onset oftherapeutic benefit, increase onset of coagulant activity, and/or anenhanced therapeutic index. Thus, provided herein are modified FVIIpolypeptides containing a modification at amino acid position 286 bymature FVII numbering that exhibit increased coagulation activitycompared to an unmodified FVII polypeptide. Such modified FVIIpolypeptides can be used in the treatment of bleeding disorders orevents, such as hemophilias, surgery, trauma, and injury, where FVIIpolypeptides can function to promote blood coagulation. Because of anincreased coagulant activity, the modified FVII polypeptides providedherein that contain a modification at amino acid position 286 by matureFVII numbering offer advantages over treatment with a wild-type FVIIpolypeptide, such as NovoSeven® Factor VII, including a decrease in theamount of administered FVII that is required to maintain a sufficientconcentration of active FVII in the serum for hemostasis. This can leadto, for example, lower doses and/or dosage frequency necessary toachieve comparable biological effects, faster onset of therapeuticbenefit, longer duration of action, higher comfort and acceptance bysubjects, and/or attenuation of undesired secondary effects.

i. Basic Amino Acid Substitutions at Position 286

Provided are modified FVII polypeptides in which the glutamine atposition 286 (numbering corresponding the mature FVII polypeptide setforth in SEQ ID NO:3; corresponding to position 143 by chymotrypsinnumbering) is replaced with a basic amino acid residue, such as any oneof arginine (Arg, R), histidine (His, H) or lysine (Lys, K). Inparticular, provided herein are modified FVII polypeptides in which theglutamine at position 286 is replaced with an arginine (i.e. Q286R,corresponding to Q143R by chymotrypsin numbering). Modeling studiesindicate that substitution of the glutamine with an arginine results inthe loss of two key interactions that stabilize an inactive conformationof the FVIIa oxyanion hole in wild-type or unmodified FVII. Thedestabilizing interactions in the wild-type or unmodified FVIIpolypeptide include the interaction between the sidechain of Q286(corresponding to Q143 by chymotrypsin numbering) and the mainchainamide of G342 (corresponding to G193 by chymotrypsin numbering), and theinteraction between the mainchain carbonyl of K341 (corresponding to K1by chymotrypsin numbering) and the mainchain amide of S195(corresponding to S344 by chymotrypsin numbering). By substituting thewild-type glutamine with an arginine at position 286, however, not onlyare these interactions lost, but two important new interactions arecreated. These include the creation of a salt bride between the basicsidechain of the modified amino acid R286 (R143 by chymotrypsinnumbering) and the acidic sidechain of the native D289 (D146 bychymotrypsin numbering), and an interaction of the mainchain amide ofthe modified amino acid R286 and the mainchain carbonyl of K341 thatstabilize an active conformation of the modified FVIIa polypeptide.Additionally, the new salt bridge between the modified amino acid R286and D289 is expected to alter the conformation and/or flexibility of the“autolysis loop,” which forms part of the active site cleft. Theautolysis loop is involved in determining the macromolecular substrateand inhibitor specificity of coagulation proteases. Thus, an alteredconformation and/or flexibility of this loop can result, for example, inincreased catalytic activity for the substrate (e.g. factor X and/orfactor IX) and increased resistance to inhibitors (e.g. TFPI and/orAT-III). Thus, modification of the glutamine at position 286 with abasic amino acid, such as arginine (Arg, R), histidine (His, H) orlysine (Lys, K), can result in increased catalytic and coagulantactivity compared with the wild-type FVII polypeptide. Hence, providedherein are FVII polypeptides containing a Q286R, Q286K or Q286H mutationby mature FVII numbering (corresponding to Q143R, Q143K or Q143H,respectively, by chymotrypsin numbering). Exemplary of such polypeptidesare those with a sequence of amino acids set forth in SEQ ID NOS:118,119 and 129, respectively.

Amino acid replacement of the glutamine (Gln, Q) with a basic amino acidresidue, in particular an arginine (Arg, R), at the amino acid positioncorresponding to amino acid position 286 of a mature FVII polypeptideset forth in SEQ ID NO:3 can be made in any FVII polypeptide, includinga precursor FVII polypeptide with a sequence set forth in SEQ ID NOS:1or 2, a mature FVII polypeptide set forth in SEQ ID NO:3, or anyspecies, allelic and modified variant, such as those described in theart, and active fragments thereof, that has 40%, 50%, 60%, 70%, 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toany of the FVII polypeptides set forth in SEQ ID NOS:1-3. For example,the Q286R mutation can be incorporated into any modified FVIIpolypeptide described in the art, including any of those describedelsewhere herein. Such modified FVII polypeptides include, but are notlimited to, a modified FVII polypeptide containing the mutation(s) M298Q(SEQ ID NO:158) see e.g. Persson et al., (2001) Proc. Nat. Acad. Sci.USA 98:13583-13588), E296V/M298Q (SEQ ID NO:343), V158E (SEQ ID NO:344),E296R/M298K (SEQ ID NO:345), K337A (SEQ ID NO:346), V158D/E296V/M298Q(SEQ ID NO:98; NN1731; see e.g., Persson et al., (2007) Art. Thromb.Vasc. Biol. 27(3): 683-689), V158D/E296V/M298Q/K337A (SEQ ID NO:347; seee.g. Lisman et al., (2003) J. Thromb. Haem. 1:2175-2178), V253N (SEQ IDNO:348; see e.g. U.S. Pat. No. 7,427,592), T106N (SEQ ID NO:349; seee.g. U.S. Pat. No. 7,427,592), T106N/V253N (SEQ ID NO:350; see e.g. U.S.Pat. No. 7,427,592), K143N/N145T (SEQ ID NO:351; U.S. Pat. No.7,442,524), R315N/V317T (SEQ ID NO:352; U.S. Pat. No. 7,442,524) orK143N/N145T/R315N/V317T (SEQ ID NO:353; U.S. Pat. No. 7,442,524). TheQ286R mutation also can be incorporated into chimeric FVII polypeptidesor FVII fusion polypeptides, or FVII polypeptides that are otherwisemodified, such as by glycoPEGylation (see e.g. WO2007022512, Ghosh etal., (2007) transcript of presentation at the Am. Society. Hematol.Meeting, Dec. 10, 2007). In one example, amino acid replacement of theglutamine with an arginine at the amino acid position corresponding toamino acid position 286 of a mature FVII polypeptide set forth in SEQ IDNO:3 results in a FVII polypeptide with a sequence of amino acids setforth in SEQ ID NO:118.

Provided herein are modified FVII polypeptides that contain the aminoacid substitution Q286R by mature FVII numbering (corresponding to Q143Rby chymotrypsin numbering), wherein the modified FVII polypeptidesexhibit increased coagulant activity. Such modified FVII polypeptidescan contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 30, 40, 50 or more modified positions, wherein one of themodified positions is amino acid position 286. Thus, provided herein aremodified FVII polypeptides containing two or more modifications, whereinone modification is the amino acid substitution Q286R (by mature FVIInumbering) and the modified FVII polypeptide exhibits increasedcoagulant activity compared to an unmodified FVII polypeptide. The Q286Rmutation can be combined with any other mutation described herein orknown in the art. Typically, the resulting modified polypeptide displaysincreased coagulant activity. One of skill in the art can determine thecoagulant activity of a FVII polypeptide containing the Q286Rmodification using in vitro and in vivo assays well known in the art anddescribed herein. The modified FVII polypeptides provided herein includethose that contain the Q286R mutation and also contain one or moremutations that, for example, increase resistance to antithrombin-III,increase activation of FX, increase activation of FIX, increase bindingand/or affinity to phospholipids, increase affinity for tissue factor,increase intrinsic activity, increase TF-dependent activity, alters theconformation of the polypeptide to alter zymogenicity, increasecatalytic or coagulant activity, such as by shifting the equilibriumbetween highly active and less active FVIIa conformations in favor ofthe highly active conformations, increase resistance to proteases,decrease glycosylation, increase glycosylation, reduce immunogenicity,increase stability, and/or facilitate chemical group linkage.

The increased coagulant activity of modified FVII polypeptidescontaining the amino acid substitution Q286R can be a result of anincrease in catalytic activity. The increased catalytic activity can beobserved in the presence and/or absence of tissue factor (TF). Thus, theincreased catalytic activity can be TF-dependent and/or TF-independent.The catalytic activity of a modified FVII polypeptide containing theQ286R mutation can be assessed using in vitro assays, such as the assaysdescribed in Examples 4 and 7. Such assays can determine the catalyticactivity of a modified FVII polypeptide for a substrate, such as factorX, in the presence or absence of tissue factor. Modified FVIIpolypeptides containing the Q286R mutations can exhibit increasedcatalytic activity of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, ormore in the presence and/or absence of tissue factor compared to thecatalytic activity of unmodified or wild-type FVII polypeptide either invivo or in vitro. For example, as demonstrated in Example 4, a FVIIapolypeptide containing the Q286R mutation (Q143R by chymotrypsinnumbering) as the sole modification can exhibit catalytic activity forFX in the presence or absence of TF that is approximately two to fourtimes greater than the catalytic activity exhibited by wild-type FVII.In other examples, a FVIIa polypeptide containing the Q286R and M298Qmutations can exhibit catalytic activity for FX in the presence of TFthat is approximately three to four times greater than the catalyticactivity exhibited by wild-type FVII, and can exhibit catalytic activityfor FX in the abesence of TF that is approximately seven to twenty-sixtimes greater than the catalytic activity exhibited by wild-type FVII.

Non-limiting examples of modified FVII polypeptides containing two ormore modifications, wherein one modification is the amino acidsubstitution Q286R (by mature FVII numbering) and the modified FVIIpolypeptide exhibits increased catalytic activity toward FX in thepresence and/or absence of tissue factor compared to an unmodified FVIIpolypeptide, are set forth in Table 5 and in Example 4, below. Thesequence identifier (SEQ ID NO) is identified in which exemplary aminoacid sequences of the modified FVII polypeptide are set forth. Asdiscussed in greater detail in section D.6, below, the “Gla swap FIX”modification involves deletion of the endogenous FVII Gla domain bydeleting amino acid residues A1 to Y44 (residues corresponding to amature FVII polypeptide set forth in SEQ ID NO:3) and insertion of 45amino acid residues that correspond to amino acid residues Y1 to Y45 ofthe FIX Gla domain set forth in SEQ ID NO:83. In some examples, theheterologous FIX Gla domain in the “Gla swap FIX”-modified FVIIpolypeptide contains one or more amino acid substitutions at amino acidpositions corresponding to M19, E40, K43 and/or Q44 of the FIX Gladomain set forth in SEQ ID NO:83. Such substitutions are denoted bycurly brackets (e.g. {Gla swap FIX/Q44S}). In instances where a modifiedamino acid position does not have a corresponding chymotrypsin number(i.e. is not within amino acid positions 153 to 406 corresponding to amature FVII polypeptide set forth in SEQ ID NO:3, and is not set forthin Table 1, above), the position is denoted in brackets using matureFVII numbering. For example, T158N does not have a correspondingchymotrypsin number and is set forth as T[158]N when referring tochymotrypsin numbering.

TABLE 5 Modification - mature FVII Modification - chymotrypsin SEQ IDnumbering numbering NO Gla Swap FIX/Q286R Gla Swap FIX/Q143R 131Q286R/H257A H117A/Q143R 132 S222A/Q286R S82A/Q143R 133 Q286R/S222A/H257AS82A/H117A/Q143R 134 Gla Swap FIX/S222A/Q286R S82A/Gla Swap FIX/Q143R135 Gla Swap FIX/H257A/Q286R H117A/Gla Swap FIX/Q143R 136 Gla SwapFIX/S222A/H257A/Q286R Q143R/S82A/H117A/Gla Swap FIX 137 Q286R/M298QQ143R/M156Q 138 Q286R/M298Q/K341Q Q143R/M156Q/K192Q 139Q286R/M298Q/K199E Q143R/M156Q/K60cE 140 S222A/H257A/Q286R/M298QS82A/H117A/Q143R/M156Q 150 A175S/Q286R/Q366V A39S/Q143R/Q217V 144S222A/Q286R/Q366V S82A/Q143R/Q217V 145 H257S/Q286R H117S/Q143R 146H257S/Q286R/Q366V H117S/Q143R/Q217V 147 S222A/H257A/Q286R/Q366VS82A/H117A/Q143R/Q217V 148 Q286R/H373A Q143R/H224A 149 Q286R/K341DQ143R/K192D 151 Q286R/Q366D Q143R/Q217D 152 Q286R/Q366N Q143R/Q217N 153Q286R/M298Q/Q366N Q143R/M156Q/Q217N 155 Q286R/H373F Q143R/H224F 156Q286R/M298Q/H373F Q143R/M156Q/H224F 157 Q286R/M298Q Q143R/M156Q 138T128N/P129A/Q286R T[128]N/P[129]A/Q143R 279 Gla swapFIX/T128N/P129A/S222A/Q286R Gla swap FIX/T[128]N/P[129]A/S82A/Q143R 285Gla swap FIX/S52A/S60A/S222A/Q286R Gla swap FIX/S[52]A/S[60]A/S82A/Q143R292 Gla swap FIX/Q286R/M298Q Gla swap FIX/Q143R/M156Q 141T128N/P129A/Q286R/M298Q T[128]N/P[129]A/Q143R/M156Q 280 Gla swapFIX/T128N/P129A/Q286R/M298Q Gla swap FIX/T[128]N/P[129]A/Q143R/M156Q 286{Gla swap FIX/E40L}/Q286R/M298Q {Gla swap FIX/E[40]L}/Q143R/M156Q 274{Gla swap FIX/K43I}/Q286R/M298Q {Gla swap FIX/K[43]I}/Q143R/M156Q 275{Gla swap FIX/Q44S}/Q286R/M298Q {Gla swap FIX/Q[44]S}/Q143R/M156Q 276{Gla swap FIX/M19K}/Q286R/M298Q {Gla swap FIX/M[19]K}/Q143R/M156Q 277S52A/S60A/Q286R/M298Q S[52]A/S[60]A/Q143R/M156Q 293T128N/P129A/S222A/H257A/Q286R/M298QT[128]N/P[129]A/S82A/H117A/Q143R/M156Q 287S52A/S60A/S222A/H257A/Q286R/M298Q S[52]A/S[60]A/S82A/H117A/Q143R/M156Q298 T128N/P129A/Q286R/H373F T[128]N/P[129]A/Q143R/H224F 281S52A/S60A/Q286R/H373F S[52]A/S[60]A/Q143R/H224F 296T128N/P129A/Q286R/M298Q/H373F T[128]N/P[129]A/Q143R/M156Q/H224F 288S52A/S60A/Q286R/M298Q/H373F S[52]A/S[60]A/Q143R/M156Q/H224F 297V21D/Q143R/E154V/M156Q V21D/Q143R/E154V/M156Q 282 Gla swapFIX/S222A/T239V/Q286R Gla swap FIX/S82A/T99V/Q143R 301 T239V/Q286R/M298QT99V/Q143R/M156Q 302 Gla swap FIX/T239V/Q286R/M298Q Gla swapFIX/T99V/Q143R/M156Q 304 S222A/T239V/H257A/Q286R/M298QS82A/T99V/H117A/Q143R/M156Q 303 T239V/Q286R/H373F T99V/Q143R/H224F 305T239V/Q286R/M298Q/H373F T99V/Q143R/M156Q/H224F 306 T239I/Q286RT99I/Q143R 308 GlaSwapFIX/S222A/T239I/Q286R Gla swap FIX/S82A/T99I/Q143R310 T239I/Q286R/M298Q T99I/Q143R/M156Q 311 Gla swapFIX/T239I/Q286R/M298Q Gla swap FIX/T99I/Q143R/M156Q 313S222A/T239I/H257A/Q286R/M298Q S82A/T99I/H117A/Q143R/M156Q 312T239I/Q286R/H373F T99I/Q143R/H224F 314 T239V/Q286R T99V/Q143R 299T239I/Q286R/M298Q/H373F T99I/Q143R/M156Q/H224F 315 H257S/Q286R/M298QH117S/Q143R/M156Q 322 Gla swap FIX/Q286R/S222A/H257S Gla swapFIX/Q143R/S82A/H117S 321 S222A/H257S/Q286R/M298Q S82A/H117S/Q143R/M156Q324 H257S/Q286R/M298Q/H373F H117S/Q143R/M156Q/H224F 325S222A/Q286R/M298Q/H373F S82A/Q143R/M156Q/H224F 326 Gla swapFIX/S222A/Q286R/M298Q/H373F Gla swap FIX S82A/Q143R/M156Q/H224F 318S222A/Q286R/M298Q S82A/Q143R/M156Q 328 Gla swap FIX/S222A/Q286R/M298QGla swap FIX S82A/Q143R/M156Q 317 Gla swap FIX/S222A/Q286R/H373F Glaswap FIX/S82A/Q143R/H224F 316 H257A/Q286R/M298Q H117A/Q143R/M156Q 321T128N/P129A/A175S/Q286R/M298Q T[128]N/P[129]A/A39S/Q143R/M156Q 337A122N/G124S/A175S/Q286R/M298Q A[122]N/G[124]S/A39S/Q143R/M156Q 338T128N/P129A/A175S/S222A/H257A/Q286R/M298QT[128]N/P[129]A/A39S/S82A/H117A/Q143R/M156Q 339A122N/G124S/A175S/S222A/H257A/Q286R/M298QA[122]N/G[124]S/A39S/S82A/H117A/Q143R/M156Q 340T128N/P129A/A175S/Q286R/M298Q/H373FT[128]N/P[129]A/A39S/Q143R/M156Q/H224F 341A122N/G124S/A175S/Q286R/M298Q/H373FA[122]N/G[124]S/A39S/Q143R/M156Q/H224F 342 V158D/Q286R/E296V/M298Q/H373FV21D/Q143R/E154V/M156Q/H224F 320 {Gla Swap FIX/K43I}/ {Gla SwapFIX/K[43]I}/ 355 T128N/P129A/Q286R/M298Q T[128]N/P[129]A/Q143R/M156QT128N/P129A/Q286R/M298Q/Q366N T[128]N/P[129]A/Q143R/M156Q/Q217N 356 {GlaSwap FIX/K43I}/Q286R/M298Q/Q366N {Gla Swap FIX/K[43]I}/Q143R/M156QQ217N357 {Gla Swap FIX/K43I}/ {Gla Swap FIX/K[43]I}/ 358T128N/P129A/Q286R/M298Q/Q366N T[128]N/P[129]A/Q143R/M156QQ217NV158D/Q286R/E296V/M298Q V21D/Q143R/E154V/M156Q 360T128N/P129A/Q286R/M298Q/Q366N/H373FT[128]N/P[129]A/Q143R/M156Q/Q217N/H224F 364 T239V/Q286R/M298Q/Q366NT99V/Q143R/M156Q/Q217N 365 T239I/Q286R/M298Q/Q366NT99I/Q143R/M156Q/Q217N 366 T128N/P129A/T239V/Q286R/M298QT[128]N/P[129]A/T99V/Q143R/M156Q 367T128N/P129A/S222A/T239V/H257A/Q286R/M298QT[128]N/P[129]A/S82A/T99V/H117A/Q143R/M156Q 368T128N/P129A/T239V/Q286R/M298Q/H373FT[128]N/P[129]A/T99V/Q143R/M156Q/H224F 369 T128N/P129A/T239I/Q286R/M298QT[128]N/P[129]A/T99I/Q143R/M156Q 370 T128N/P129A/T239I/Q286R/M298Q/H373FT[128]N/P[129]A/T99I/Q143R/M156Q/H224F 371

A FVII polypeptide containing the Q286R mutation by mature FVIInumbering also can exhibit increased resistance to AT-III. The increasedresistance to AT-III can be a result of a decreased rate of inhibitionby AT-III or decreased binding to AT-III under specified conditions,such as following injection into an animal or patient. Resistance toAT-III can be demonstrated by measuring the second order rate constantfor inhibition of wild type and variant FVIIa polypeptides. Other invitro methods, such as BIAcore® assays, can also be used. The modifiedFVII polypeptides can exhibit increased resistance to the inhibitoryeffects of AT-III compared to an unmodified FVII polypeptide, which canbe assessed in in vitro assays such as those described in Example 5.Modified FVII polypeptides containing the Q286R mutations can exhibitincreased resistance to AT-III of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%,500%, or more compared to the resistance to AT-III of unmodified orwild-type FVII polypeptide either in vivo or in vitro. For example, asdemonstrated in Example 5 below, a FVIIa polypeptide containing theQ286R mutation (Q143R by chymotrypsin numbering) can exhibit catalyticactivity for FX in the presence of AT-III and the absence of TF that istwo to four times or more greater than the catalytic activity exhibitedby wild-type FVII. Thus, the modified Q286R FVII polypeptide can exhibitan increase in resistance to AT-III of about 200% to 400% of that of anunmodified FVII polypeptide.

Increased catalytic activity and increased resistance to AT-III canmanifest as increased coagulant activity in the presence and/or absenceof TF. Such activities can be assessed in vitro, ex vivo or in vivo,such as by administration to a human or animal subject. The coagulationactivity of the modified FVII polypeptides containing the Q286R mutationcan be increased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%,500%, or more compared to the coagulation activity of unmodified orwild-type FVII polypeptide either in vivo or in vitro. For example,Example 6.B.2 demonstrates that a FVIIa polypeptide containing the Q286Rmutation (Q143R by chymotrypsin numbering) exhibits coagulation activityin a mouse bleeding model that is greater (approximately 2 fold) thanthe coagulation activity exhibited by a wild-type FVII polypeptide (e.g.NovoSeven® FVII). FVIIa polypeptide containing the Q286R and M298Qmutations (Q143R and M156Q, respectively, by chymotrypsin numbering)exhibit even greater coagulation activity.

ii. Other Mutations at Position 286

The glutamine at the amino acid position corresponding to position 286of the FVII polypeptide set forth in SEQ ID NO:3 can be replaced with anamino acid other than a basic amino acid (i.e. other than arginine,histidine or lysine). Such substitutions can alter the conformation ofthe oxyanion hole, for example, resulting in a conformation thatincreases the catalytic activity of the modified FVII polypeptidecompared to a wildtype FVII polypeptide. Modified FVII polypeptides thathave an altered oxyanion hole conformation can exhibit increasedcatalytic activity of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, ormore compared to the catalytic activity of unmodified or wild-type FVIIpolypeptide when measured using either in vivo, ex vivo, or in vitroassays.

Table 6 provides non-limiting examples of exemplary amino acidreplacements at Q286 other than replacement with arginine, correspondingto amino acid positions of a mature FVII polypeptide as set forth in SEQID NO:3. As noted, such FVII polypeptides are designed to change theconformation of the oxyanion hole to a more effective conformation, andtherefore have increased coagulant activity. In reference to suchmutations, the first amino acid (one-letter abbreviation) corresponds tothe amino acid that is replaced, the number corresponds to the positionin the mature FVII polypeptide sequence with reference to SEQ ID NO:3,and the second amino acid (one-letter abbreviation) corresponds to theamino acid selected that replaces the first amino acid at that position.The amino acid positions for mutation also are referred to by thechymotrypsin numbering scheme. In Table 6 below, the sequence identifier(SEQ ID NO) is identified in which exemplary amino acid sequences of themodified FVII polypeptide are set forth.

TABLE 6 Modification - mature FVII Modification - chymotrypsin SEQ IDnumbering numbering NO Q286N Q143N 113 Q286E Q143E 114 Q286D Q143D 115Q286S Q143S 116 Q286T Q143T 117 Q286A Q143A 120 Q286V Q143V 121 Q286MQ143M 122 Q286L Q143L 123 Q286Y Q143Y 124 Q286G Q143G 125 Q286F Q143F126 Q286I Q143I 127 Q286P Q143P 128 Q286W Q143W 130

Modified FVII polypeptides that have an altered oxyanion holeconformation can exhibit increased catalytic activity of about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 200%, 300%, 400%, 500%, or more compared to the catalytic activityof unmodified or wild-type FVII polypeptide when measured using eitherin vivo, ex vivo, or in vitro assays. In some examples, the modifiedFVII polypeptides that have an altered oxyanion hole conformation alsocan exhibit increased resistance to endogenous protease inhibitors(i.e., decreased rate of inhibition by or decreased affinity forinhibitors) such as TFPI or AT-III by about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%,400%, 500%, or more compared to the rate of inhibition by or affinityfor endogenous inhibitors exhibited by unmodified or wild-type FVIIpolypeptide either in vivo, ex vivo, or in vitro. Increased catalyticactivity and/or resistance to endogenous inhibitors such as AT-IIIresistance of such modified FVII polypeptides also can be manifested asincreased coagulation activity, duration of coagulant activity, fasterinitiation of coagulant activity and/or enhanced therapeutic index. Forexample, the coagulation activity of the modified FVII polypeptides canbe increased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, ormore compared to the coagulation activity of unmodified or wild-typeFVII polypeptide either in vivo, ex vivo, or in vitro.

2. Increased Resistance to AT-III

Antithrombin III (also known as antithrombin or AT-III) is an importantanticoagulant serpin (serine protease inhibitor). AT-III is synthesizedas a precursor protein containing 464 amino acid residues (SEQ IDNO:122). In the course of secretion a 32 residue signal peptide iscleaved to generate a 432 amino acid mature human antithrombin (SEQ IDNO:123). The 58 kDa AT-III glycoprotein circulates in the blood andfunctions as a serine protease inhibitor (serpin) to inhibit a largenumber of serine proteases of the coagulation system. The principaltargets of AT-III are thrombin and factor Xa, although AT-III also hasbeen shown to inhibit the activities of FIXa, FXIa, FXIIa and, to alesser extent, FVIIa. The action of AT-III is greatly enhanced byglycosaminoglycans, such as the naturally occurring heparan sulphate orthe various tissue-derived heparins that are widely used asanticoagulants in clinical practice. AT-III binds in a highly specificmanner to a unique pentasaccharide sequence in heparin that induces aconformational change in the reactive center loop. In such aconformation, the reactive center loop of AT-III can more efficientlyinteract with the reactive site of the serine protease, and effectinhibition.

AT-III is not normally inhibitory to free plasma FVIIa, even in thepresence of heparin, likely due to the zymogen-like conformation ofFVIIa that prevents efficient interaction with AT-III. The inhibitoryeffects of AT-III do increase, however, once FVIIa complexes with TF.Binding of AT-III to the TF/FVIIa complex can release FVIIa from TF andmaintains it in an inactive complex with AT-III. The increased affinityof AT-III for TF-bound FVIIa compared with FVIIa alone presumablyreflects the maturation of the active site of FVIIa when it is complexedwith TF, therefore making it amenable to AT-III binding (Rao et al.(1993) Blood 81:2600-2607). Thus, the impact of AT-III on FVIIa isproportional to the intrinsic activity of the FVIIa molecule itself,unless mutations have been added to the FVIIa polypeptide that mediateresistance to AT-III. While FVIIa retains its zymogen-like conformation,AT-III has little effect. If, however, FVIIa changes conformation to amore active form, such as by binding TF, or by specific in vitromodifications, AT-III inhibition increases significantly. FVIIapolypeptides that are modified to have increased intrinsic activityoften display simultaneous increases in susceptibility to AT-IIIinhibition. For example, modification of one or more amino acids in theactivation pocket of FVIIa, such as by amino acid replacementscorresponding to K337A, L305V, M298Q, V158D and E296V substitutions(relative to the mature FVII sequence set forth in SEQ ID NO:3), resultsin increased sensitivity of the FVIIa polypeptide to AT-III, therebyinhibiting FVIIa activity by up to 90% (Persson et al. (2001) PNAS98:13583-13588). In another example, induction of a more zymogen-likeconformation by modification of amino acids involved in the α-helix ofFVIIa, while increasing the activity of the modified FVIIa protein, alsoincreases its susceptibility to AT-III (Persson et al. (2004) Biochem J379:497-503).

Exemplary Modifications to Effect Increased Resistance to AT-III

Modifications can be made to a FVII polypeptide that increase itsresistance to AT-III. Generally, such modified FVII polypeptides retainat least one activity of a FVII polypeptide. Typically, suchmodifications include one or more amino acid substitutions at anyposition of the FVII polypeptide that are involved in the interaction ofFVIIa with AT-III. Such modifications can, for example, result inreduced binding of the modified FVII to AT-III. The modified FVIIpolypeptides are therefore resistant to the naturally inhibitory effectsof AT-III with respect to coagulation initiation. When evaluated in anappropriate in vitro assay, or in vivo, such as following administrationto a subject as a pro-coagulant therapeutic, the modifiedAT-III-resistant FVII polypeptides display increased coagulant activityas compared with unmodified FVII polypeptides.

As described herein below, one of skill in the art can empirically orrationally design modified FVII polypeptides that display increasedresistance to AT-III. Such modified FVII polypeptides can be tested inassays known to one of skill in the art to determine if such modifiedFVII polypeptides display increased resistance to AT-111. For example,such modified AT-III polypeptides can be tested for binding to AT-III.Generally, a modified FVII polypeptide that has increased resistance toAT-III will exhibit decreased binding and/or decreased affinity forAT-III. Typically, such assays are performed on a two-chain form ofFVII, such as the activated form of FVII (FVIIa). Further, assays todetermine effects of AT-III are generally performed in the presence ofheparin and the presence of tissue factor, although such assays also canbe performed in the absence of one or both cofactors.

Provided herein are modified FVII polypeptides exhibiting increasedresistance to AT-III. Resistance to inhibition by ATIII is relevant bothin the presence and absence of TF. FVII polypeptide variants providedherein have been modified at one or more of amino acid positions 239,931, 366 and 373 (corresponding to amino acid positions 99, 170i, 217and 224, respectively, by chymotrypsin numbering). These amino acidresidues can be modified such as by amino acid replacement, deletion orsubstitution. The identified residues can be replaced or substitutedwith any another amino acid. Alternatively, amino acid insertions can beused to alter the conformation of a targeted amino acid residue or theprotein structure in the vicinity of a targeted amino acid residue.

Any amino acid residue can be substituted for the endogenous amino acidresidue at the identified positions. Typically, the replacement aminoacid is chosen such that it interferes with the interaction between FVIIand AT-III. In some examples, the threonine residue at position 239(corresponding to position 99 by chymotrypsin numbering) is replacedwith a serine (Ser, S), asparagine (Asn, N), glutamine (Gln, Q), valine(Val, V), leucine (Leu, L), histidine (His, H), or isoleucine (Ile, I).In other examples, the proline at position 321 (corresponding toposition 170i by chymotrypsin numbering) is replaced with a lysine (Lys,K), glutamic acid (Glu, E), serine (Ser, S), or tyrosine (Tyr, Y). Infurther examples, the glutamine at position 366 (corresponding toposition 217 by chymotrypsin numbering) is replaced with an asparagine(Asn, N), aspartic acid (Asp, D), glutamic acid (Glu, E), serine (Ser,S), threonine (Thr, T), lysine (Lys, K), or valine (Val, V). In otherexamples, the histidine at position 373 (corresponding to position 224by chymotrypsin numbering) is replaced with an aspartic acid (Asp, D),glutamic acid (Glu, E), serine (Ser, S), phenylalanine (Phe, F) oralanine (Ala, A). In a further embodiment, combination mutants can begenerated. Included among such combination mutants are those having twoor more mutations of the residues T239, P321, Q366 and H373(corresponding to T99, P170i, Q217 and H224, respectively, bychymotrypsin numbering). For example, a modified FVII polypeptide canpossess amino acid substitutions at 2, 3, 4 or 5 of the identifiedpositions. Hence, a modified polypeptide can display 1, 2, 3, 4 or 5mutations that can result in increased resistance of the modified FVIIpolypeptide to the inhibitory effects of AT-III. For example, a FVIIpolypeptide can be modified at amino acid position 366 and amino acidposition 373. In some example, the positions are modified by amino acidreplacement, such as, for example, replacement of the glutamine atposition 366 with an aspartic acid, and replacement of the histidine atposition 373 with a glutamic acid.

Table 7 provides non-limiting examples of exemplary amino acidreplacements at the identified residues, corresponding to amino acidpositions of a mature FVII polypeptide as set forth in SEQ ID NO:3.Included amongst these are exemplary combination mutations. As noted,such FVII polypeptides are designed to increase resistance to AT-III andtherefore have increased coagulant activity. In reference to suchmutations, the first amino acid (one-letter abbreviation) corresponds tothe amino acid that is replaced, the number corresponds to the positionin the mature FVII polypeptide sequence with reference to SEQ ID NO:3,and the second amino acid (one-letter abbreviation) corresponds to theamino acid selected that replaces the first amino acid at that position.The amino acid positions for mutation also are referred to by thechymotrypsin numbering scheme. In Table 7 below, the sequence identifier(SEQ ID NO) is identified in which exemplary amino acid sequences of themodified FVII polypeptide are set forth.

TABLE 7 Modification - mature FVII Modification - chymotrypsin SEQ IDnumbering numbering NO T239S T99S 159 T239N T99N 160 T239Q T99Q 161T239V T99V 162 T239L T99L 163 T239H T99H 164 T239I T99I 165 P321K P170iK166 P321E P170iE 167 P321Y P170iY 168 P321S P170iS 169 Q366D Q217D 170Q366E Q217E 171 Q366N Q217N 172 Q366T Q217T 173 Q366S Q217S 174 Q366VQ217V 175 Q366I Q217I 176 Q366L Q217L 177 Q366M Q217M 178 H373D H224D179 H373E H224E 180 H373S H224S 181 H373F H224F 182 H373A H224A 183Q366D/H373E Q217D/H224E 184 Q366V/H373V Q217V/H224V 185 Q366V/H373LQ217V/H224L 186 Q366V/H373I Q217V/H224I 187

Modified FVII polypeptides that have increased resistance for AT-III canexhibit a reduction in the extent of inhibition under specifiedconditions or in the second order rate constant for inhibition by AT-IIIby at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or morecompared to the extent of inhibition or the second order rate constantfor inhibition of unmodified or wild-type FVII polypeptide either invivo or in vitro. Thus, the modified FVII polypeptides can exhibitincreased resistance to AT-III that is at least or about 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,200%, 300%, 400%, 500%, or more of the resistance exhibited by anunmodified FVII polypeptide. Increased resistance to AT-III by suchmodified FVII polypeptides also can be manifested as increasedcoagulation activity, duration of coagulation activity and/or enhancedtherapeutic index in the presence of AT-III. The coagulation activity ofthe AT-III-modified FVII polypeptides can be increased by at least about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to thecoagulation activity of unmodified or wild-type FVII polypeptide eitherin vivo or in vitro.

3. Increased Resistance to Inhibition by Zn²⁺

The amidolytic activity of FVIIa is regulated by allosteric alterationsinduced by binding of calcium ions and tissue factor (TF). Free FVIItypically exists in an inactive conformation. Binding to Ca²⁺ and TFinduces a change in conformation and increased amidolytic activity(Pederson et al., (1990) J. Biol. Chem. 265:16786-16793). In contrast,the binding of zinc ions to FVIIa has been shown to have an inhibitoryeffect on activity. Binding of Zn²⁺ to FVIIa results in decreasedamidolytic activity and reduced affinity for TF. Studies indicate thatCa²⁺ and Zn²⁺ compete for binding to FVIIa, such that in the presence ofCa²⁺, the inhibitory effect of Zn²⁺ is reduced. Furthermore, FVIIa boundto TF is less susceptible to zinc inhibition.

In addition to the Zn²⁺ binding sites in the Gla domain, the binding ofwhich does not affect FVIIa amidolytic activity, two Zn²⁺ binding siteshave been mapped to the protease domain of FVII (Petersen et al., (2000)Protein Sci. 9:859-866, Bajaj et al., (2006) J. Biol. Chem.281:24873-24888). Mapping of these binding sites in the protease domainindicates that the first Zn²⁺ binding site involves the side chains ofamino acid residues H216, E220 and S222 (H76, E80 and S82 bychymotrypsin numbering), and the second Zn²⁺ binding site involves theside chains of amino acid residues H257, D219 and K161 (H117, D79 andK24 by chymotrypsin numbering).

Zn²⁺ could, therefore, have a physiologic role in regulating homeostasisas a FVII inhibitor. It has been postulated that these inhibitoryeffects occur as a result of an increase in Zn²⁺ concentration at thesite of the clot following platelet activation (Bajaj et al., (2006) J.Biol. Chem. 281:24873-24888). Platelets store large amounts of Zn²⁺ inthe cytoplasm and α-granules, which are released upon plateletactivation. This could increase the local concentration of Zn²⁺ which inturn could inhibit FVIIa activity and FVIIa binding to TF.

Exemplary Modifications to Increase Resistance to Inhibition by Zn²⁺

Provided herein are modified FVII polypeptides exhibiting increasedresistance to the inhibitory effects of Zn²⁺. This can be achieved, forexample, by mutation of one or more residues in FVII involved in theinteraction and binding with Zn²⁺ to reduce or prevent such binding,thereby making the modified FVII polypeptides resistant to theinhibitory effects of Zn²⁺ with respect to catalytic activity and TFbinding. When evaluated in an appropriate in vitro assay, or in vivo,such as following administration to a subject as a pro-coagulanttherapeutic, the modified FVII polypeptides can display increasedcoagulant activity as compared with unmodified FVII polypeptides.

Provided herein are modified FVII polypeptides having one or moremutations in residues that may be involved in Zn²⁺ binding in theprotease domain. Such residues include, but are not limited to, K161,H216, D219, E220, S222 and H257, with numbering relative to the aminoacid positions of a mature FVII polypeptide set forth in SEQ ID NO:3(corresponding to K24, H76, D79, E80, S82 and H117, respectively, bychymotrypsin numbering). In some examples, one or more of the amino acidresidues H216, S222 and H257 (corresponding to H76, S82 and H117,respectively, by chymotrypsin numbering) are modified, such as by aminoacid replacement or deletion. Any amino acid residue can be used toreplace the endogenous residue at the identified positions. For example,provided herein are modified FVII polypeptides in which the histidine atamino acid position 216 is replaced with a serine, alanine, lysine orarginine residue. In another example, the serine at amino acid position222 is replaced with an alanine or lysine residue, or the histidine atposition 257 is replaced with an alanine or serine residue. In a furtherembodiment, the lysine at position 161 is replaced with a serine,alanine or valine residue. Modifications also include amino acidinsertions at or near the amino acid positions identified as beinginvolved in Zn²⁺ binding. Such insertions can disrupt the Zn²⁺ bindingsite, resulting in a modified FVII polypeptide with decreased binding toZn²⁺.

Combination mutants in which amino acid replacements are made at morethan one of the above-identified residues in a FVII polypeptide also canbe generated. Included among such combination mutants are those havingtwo or more mutations of the residues K161, H216, D219, E220, S222 andH257 (corresponding to K24, H76, D79, E80, S82 and H117, respectively,by chymotrypsin numbering). For example, a modified FVII polypeptide canpossess amino acid substitutions at 2, 3, 4, 5 or 6 of the identifiedpositions. Hence, a modified polypeptide can display 1, 2, 3, 4, 5 or 6mutations that can result in decreased ability of the modified FVIIpolypeptide to bind Zn²⁺. For example, a FVII polypeptide can bemodified by amino acid replacement of the serine at position 222 with alysine, and the histidine at position 257 with an alanine residue.

The modified FVII polypeptides that have increased resistance to theinhibitory effects of Zn²⁺ can exhibit an increase by at least about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200%, 300%, 400%, 500%, or more compared to the resistance ofunmodified or wild-type FVII polypeptide either in vivo or in vitro. Areduction in Zn²⁺ binding and, therefore, increased resistance againstthe inhibitory effects of Zn²⁺, by such modified FVII polypeptides alsocan be manifested as increased coagulation activity in the presence ofZn²⁺. The coagulation activity of the modified FVII polypeptides can beincreased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, ormore compared to the coagulation activity of unmodified or wild-typeFVII polypeptide either in vivo or in vitro.

Table 8 provides non-limiting examples of exemplary amino acidreplacements at the identified residues, corresponding to amino acidpositions of a mature FVII polypeptide as set forth in SEQ ID NO:3.Included amongst these are exemplary combination mutations. As noted,such FVII polypeptides are designed to exhibit reduced ability to bindZn²⁺ and, therefore, increased resistance against the inhibitory effectsof Zn²⁺. Thus, the modified FVII polypeptide can have increasedcoagulant activity. In reference to such mutations, the first amino acid(one-letter abbreviation) corresponds to the amino acid that isreplaced, the number corresponds to the position in the mature FVIIpolypeptide sequence with reference to SEQ ID NO:3, and the second aminoacid (one-letter abbreviation) corresponds to the amino acid selectedthat replaces the first amino acid at that position. The amino acidpositions for mutation also are referred to by the chymotrypsinnumbering scheme. In Table 8 below, the sequence identifier (SEQ ID NO)is identified in which exemplary amino acid sequences of the modifiedFVII polypeptide are set forth.

TABLE 8 Modification - mature Modification - FVII chymotrypsin SEQ IDnumbering numbering NO K161S K24S 188 K161A K24A 189 K161V K24V 190H216S H76S 191 H216A H76A 192 H216K H76K 193 H216R H76R 194 S222A S82A195 S222K S82K 196 S222V S82V 197 S222N S82N 198 S222E S82E 199 S222DS82D 200 H257A H117A 201 H257S H117S 202 S222K/H257A S82K/H117A 203H216A/H257A H76A/H117A 204 H216A/S222A H76A/S82A 205

4. Altered Glycosylation

The properties and activities of a protein can be altered by modulatingthe extent, level, and/or type of glycosylation. For example,glycosylation can increase serum-half-life of polypeptides by increasingthe stability, solubility, and reducing the immunogenicity of a protein.Glycosylation can increase the stability of proteins by reducing theproteolysis of the protein and can protect the protein from thermaldegradation, exposure to denaturing agents, damage by oxygen freeradicals, and changes in pH. Glycosylation also can allow the targetprotein to evade clearance mechanisms that can involve binding to otherproteins, including cell surface receptors. Carbohydrate moieties thatcontain sialic acid can affect the solubility of a protein. The sialicacid moieties are highly hydrophilic and can shield hydrophobic residuesof the target protein. This decreases aggregation and precipitation ofthe target protein. Decreased aggregation also aids in the prevention ofthe immune response against the target protein. Carbohydrates canfurthermore shield immunogenic sequences from the immune system. Thevolume of space occupied by the carbohydrate moieties can decrease theavailable surface area that is surveyed by the immune system. Theseproperties lead to the reduction in immunogenicity of the targetprotein.

Glycosylation sites provide a site for attachment of monosaccharides andoligosaccharides to a polypeptide via a glycosidic linkage, such thatwhen the polypeptide is produced in a eukaryotic cell capable ofglycosylation, it is glycosylated. The two main types of glycosylationare N-linked glycosylation, where the sugar units are attached via theamide nitrogen of an asparagine residue, and O-linked glycosylation,where the sugar units are attached via the hydroxyl group of serine,threonine, hydroxylysine or hydroxyproline residues. Other more minorforms of glycosidic linkages include S-linkage to cysteine and C-linkageto tryptophan. N-linked glycosylation occurs at asparagines in theconsensus sequence -Asn-Xaa-Ser/Thr/Cys where Xaa is not proline. Thereis no known motif for O-glycosylation, although O-glycosylation is moreprobable in sequences with a high proportion of serine, threonine andproline residues. The presence of a potential glycosylation site doesnot, however, ensure that the site will be glycosylated duringpost-translational processing in the ER. Furthermore, the level ofglycosylation may vary at a given site, and one site may have manydifferent glycan structures. There are four naturally occurringglycosylation sites in FVII; two N-glycosylation sites at N145 and N322,and two O-glycosylation sites at S52 and S60, corresponding to aminoacid positions in the mature FVII polypeptide set forth in SEQ ID NO:3.

Exemplary Modifications to Alter Glycosylation

Provided herein are FVII polypeptides that have been modified byaltering the level and/or type of glycosylation as compared to anunmodified FVII polypeptide. Glycosylation can be increased or decreasedcompared to the unmodified FVII. In some instances, the level ofglycosylation is increased, resulting in a hyperglycosylated FVIIpolypeptide. This can be achieved, for example, by incorporation of atleast one non-native glycosylation site not found in the unmodified FVIIpolypeptide to which a carbohydrate moiety is linked. HyperglycosylatedFVII polypeptides also can be generated by linkage of a carbohydratemoiety to at least one native glycosylation site found but notglycosylated in the unmodified FVII polypeptide. In other examples, thelevel of glycosylation in a modified FVII polypeptide is decreasedcompared to an unmodified FVII polypeptide. This can be achieved byeliminating one or more native glycosylation sites, such as by aminoacid replacement or deletion. One or more of the amino acid residues atamino acid positions 52, 60, 145 and 322 corresponding to a mature FVIIpolypeptide set forth in SEQ ID NO:3 can be deleted or can be replacedwith an amino acid residue that can not be linked to carbohydratemoieties. For example, the serine residues at positions 52 and/or 60 canbe replaced with an alanine residue, thereby eliminating one or both ofthe native O-glycosylation sites. Thus, glycosylation sites in a FVIIpolypeptide can be introduced, altered, eliminated or rearranged.

A FVII polypeptide can be modified at one or more positions to alterglycosylation of the polypeptide. The modified FVII polypeptidesprovided herein that have altered glycosylation compared to anunmodified FVII polypeptide can have no glycosylation, O-linkedglycosylation, N-linked glycosylation, and/or a combination thereof. Insome examples, a modified FVII polypeptide includes 1, 2, 3, 4, 5 ormore carbohydrate moieties, each linked to different glycosylationsites. The glycosylation sites can be a native glycosylation site and/ora non-native glycosylation site. In some examples, the modified FVIIpolypeptide is glycosylated at more than one non-native glycosylationsite. For example, a modified FVII polypeptide can be modified tointroduce 1, 2, 3, 4, 5 or more non-native glycosylation sites.

Non-native glycosylation sites can be introduced by amino acidreplacement. O-glycosylation sites can be created, for example, by aminoacid replacement of a native residue with a serine or threonine.N-glycosylation sites can be created by establishing the motifAsn-Xaa-Ser/Thr/Cys, where Xaa is not proline. Creation of thisconsensus sequence by amino acid modification could involve replacementof a native amino acid residue with an asparagine, replacement of anative amino acid residue with a serine, threonine or cysteine, orreplacement of a native amino acid residue with an asparagine and aminoacid replacement of native residue with a serine, threonine or cysteine.For example, the lysine at position 109 (based on numbering of a matureFVII set forth in SEQ ID NO:3) can be replaced with an asparagine tocreate a new Asn-Xaa-Ser motif in the EGF1 domain and a newN-glycosylation site at amino acid position 109. In another example, thealanine at position 292 is replaced with an asparagine and the alanineposition 294 is replaced with a serine to create a new Asn-Xaa-Ser motifand a new N-glycosylation site at amino acid position 292. In a furtherexample, the alanine at position 175 is replaced with a serine to createa new Asn-Xaa-Ser motif at amino acid positions 173-175 based onnumbering of a mature FVII set forth in SEQ ID NO:3, and a newN-glycosylation site at amino acid position 173. Non-nativeglycosylation sites can be created in any region in the FVIIpolypeptide. For example, one or more glycosylation sites can beintroduced into the EGF1 domain, which corresponds to amino acidpositions 46-82 of the mature FVII polypeptide in SEQ ID NO:3. In otherexamples, non-native glycosylation sites are introduced into theprotease domain region of the FVII polypeptide, or in positions that canassociate with the protease domain region upon protein folding.

Native glycosylation sites can be modified to prevent glycosylation orenhance or decrease glycosylation, while other positions in the FVIIpolypeptide can be modified to introduce non-native glycosylation sites.In some examples, the carbohydrate content of the FVII polypeptide canbe modified. For example, the number position, bond strength, structureand composition of the carbohydrate linkages (i.e., structure of thecarbohydrate based on the nature of the glycosidic linkages or branchesof the carbohydrate) of carbohydrate moieties added to the FVIIpolypeptide can be altered.

The modified FVII polypeptides provided herein that have alteredglycosylation retain at least one activity of FVII. Typically, themodified FVII polypeptides provided herein that have alteredglycosylation exhibit increased coagulant activity compared to anunmodified FVII. In some examples, the level of glycosylation of a FVIIpolypeptide is increased. The level of glycosylation can be increased byat least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more comparedto the level of glycosylation of unmodified or wild-type FVIIpolypeptide. In other examples, the level of glycosylation is decreased.The level of glycosylation can be decreased by at least about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 200%, 300%, 400%, 500%, or more compared to the level ofglycosylation of unmodified or wild-type FVII polypeptide. Alteredglycosylation levels or changes in the type of glycosylation present ona modified FVII polypeptide compared to an unmodified FVII polypeptidecan be manifested as increased coagulation activity. The coagulationactivity of the modified FVII polypeptides with altered glycosylationcan be increased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%,500%, or more compared to the coagulation activity of unmodified orwild-type FVII polypeptide either in vivo or in vitro.

Table 9 provides non-limiting examples of exemplary amino acidreplacements, corresponding to amino acid positions of a mature FVIIpolypeptide as set forth in SEQ ID NO:3, that are included in a modifiedFVII polypeptide to alter glycosylation levels by adding or eliminatingglycosylation sites. The exemplary amino acid replacements can createnon-native glycosylation sites or eliminate native glycosylation sites.In some instances, two amino acid replacements are required to create anew glycosylation site. Also included in Table 9 are exemplarycombination mutations that create more than one new non-nativeglycosylation site in the FVII polypeptide. As noted above, changes inglycosylation levels can, for example, increase half-life. Thus, themodified FVII polypeptides can have increased coagulant activity. Inreference to such mutations, the first amino acid (one-letterabbreviation) corresponds to the amino acid that is replaced, the numbercorresponds to the position in the mature FVII polypeptide sequence withreference to SEQ ID NO:3, and the second amino acid (one-letterabbreviation) corresponds to the amino acid selected that replaces thefirst amino acid at that position. The amino acid positions for mutationalso are referred to by the chymotrypsin numbering scheme whereappropriate. In instances where a modified amino acid position does nothave a corresponding chymotrypsin number (i.e. is not within amino acidpositions 153 to 406 corresponding to a mature FVII polypeptide setforth in SEQ ID NO:3, and is not set forth in Table 1, above), theposition is denoted in brackets using mature FVII numbering. Forexample, A51N does not have a corresponding chymotrypsin number and isset forth as A[51]N when referring to chymotrypsin numbering. In Table 9below, the sequence identifier (SEQ ID NO) is identified in whichexemplary amino acid sequences of the modified FVII polypeptide are setforth. Also identified in Table 9 are any new non native glycosylationsite(s) generated by the modification(s).

TABLE 9 Non-native glycosylation Non-native site glycosylationModification(s) - Modification(s) - (mature site mature FVIIchymotrypsin FVII (chymotrypsin SEQ ID numbering numbering numbering)numbering NO S52A S[52]A none none 206 S60A S[60]A none none 207E394N/P395A/R396S E245N/P246A/R247S N394 N245 208 R202S R62S N200 N60d209 A292N/A294S A150N/A152S N292 N150 210 G318N G170fN N318 N170f 211A175S A39S N173 N37 212 K109N K[109]N N109 N[109] 213 A122N/G124SA[122]N/G[124]S N122 N[122] 214 A51N A[51]N N51 N[51] 215 T130N/E132ST[130]N/E[132]S N130 N[130] 216 A122N/G124S/ A[122]N/G[124]S/ N122 andN[122] and 217 E394N/P395A/R396S E245N/P246A/R247S N394 N245A122N/G124S/ A[122]N/G[124]S/ N122, N394 N[122], 218 E394N/P395A/R396S/E245N/P246A/R247S/ and N318 N245 and G318N G170fN N318 S52A/S60AS[52]A/S[60]A none none 219 S52N/P54S S[52]N/P[54]S N52 N[52] 220S119N/L121S S[119]N/L[121]S N119 N[119] 221 T128N/P129A T[128]N/P[129]AN128 N[128] 222 Q66N/Y68S Q[66]N/Y[68]S N66 N[66] 223S52N/P54S/A122N/G124S/ S[52]N/P[54]S/A[122]N/ N52, N122 N[52], 224E394N/P395A/R396S G[124]S/E245N/P246A/ and N397 N[122] and R247S N245K109N/A292N/A294S K[109]N/A150N/A152S N109 and N[109] and 225 N292 N150K109N/A175S K[109]N/A39S N109 and N[109] and 226 N173 N37S119N/L121S/A175S S[119]N/L[121]S/A39S N119 and N[119] and 271 N173 N37T128N/P129A/A175S T[128]N/P[129]A/A39A N128 and N[128] and 272 N173 N37A122N/G124S/A175S A[122]N/G[124]S/A39S N122 and N[122] and 273 N173 N37

5. Increased Binding to Serum Albumin and/or Platelet Integrin α_(IIb)β₃

Recombinant unmodified FVII has a serum half-life of only 1.5-3 hours inhumans. Increasing the serum half-life of a FVII polypeptide can reducein amount and frequency the dosages required for therapeutic effect.Several strategies can be employed to increase serum half-lifeincluding, but not limited to, increasing glycosylation, increasingprotease resistance, PEGylation and conjugation or fusion to largerproteins, such as serum albumin and the Fc portion of IgG. Suchmodifications can result in, for example, reduced degradation of theFVII polypeptide by serum proteases, reduced renal clearance, reducedhepatic clearance, and reduced neutralization or clearance by the immunesystem. Another strategy that can be employed to increase the serumhalf-life of a FVII polypeptide involves the grafting of bindingsequences into an unmodified FVII polypeptide to establish new orimproved protein-protein interactions that are not observed in anunmodified FVII polypeptide.

Binding sequences that are inserted into the unmodified FVII polypeptidecan contain about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 ormore amino acid residues that facilitate interaction with anotherprotein. The binding sequences can correspond to a binding sequencenaturally present in a native protein, or can be a synthetic bindingsequence with little or no sequence correlation to binding sequencesnaturally present in a native protein. The binding sequences used tomodify the FVII polypeptides herein specifically interact with a bindingsite on another protein, establishing a non-covalent protein-proteininteraction. In some examples, the protein for which the bindingsequence is specific is a serum protein, such as, for example, serumalbumin. Such sequences are well known in the art (see e.g.US20030069395, US20040009534, and US20070202045). In other examples, theprotein recognized by the binding sequence is a cell surface receptor orligand, such as, for example, platelet integrin α_(IIb)β₃ (Smith et al.(1995) J. Biol. Chem. 270:30486-30490). The affinity with which themodified FVII polypeptide binds to the serum protein or cell surfacereceptor is typically characterized by a dissociation constant, Kd, of 1μM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, 1 pM or less. Binding of themodified FVII polypeptide to the serum protein or cell surface receptorvia the binding sequence can reduce, for example, renal clearance orhepatic clearance of the modified FVII polypeptide compared to anunmodified FVII polypeptide. In some examples, binding of the modifiedFVII polypeptide to a cell surface receptor also can target the modifiedFVII polypeptide to a desired cell or tissue type or region in the body,thereby “concentrating” that FVII polypeptide at a particular site, suchas, for example, a blood clot. Thus, modified FVII polypeptidescontaining engrafted binding sequences can exhibit increased half-lifecompared to an unmodified FVII polypeptide.

a. Exemplary FVII Polypeptides with Serum Albumin Binding Sequences

Provided herein are modified FVII polypeptides containing serum albuminbinding sequences. The modified FVII polypeptides can bind serum albuminin vitro or in vivo, resulting in an increased half-life. Thus, providedherein are modified FVII polypeptides with increased half-life comparedto an unmodified FVII polypeptide. When evaluated in an appropriate invitro assay, or in vivo, such as following administration to a subjectas a pro-coagulant therapeutic, the modified FVII polypeptides candisplay increased coagulant activity as compared with unmodified FVIIpolypeptides.

The modified FVII polypeptides provided herein can contain serum albuminbinding sequences. The serum albumin binding sequences can be insertedwithin the unmodified FVII polypeptide or can be linked to the C- orN-terminal of the FVII polypeptide. For example, the serum albuminbinding sequence can extend from the proline residue at amino acidposition 406 at the C-terminus of the FVII polypeptide (corresponding toa mature FVII polypeptide set forth in SEQ ID NO:3). If the bindingsequences are inserted within the FVII polypeptide, insertion is at aposition such that the resulting modified FVII polypeptide retains atleast one activity of an unmodified FVII polypeptide. The bindingsequence can be inserted into the FVII polypeptide without removing anyamino acid residues in the FVII polypeptide, or can replace one or moreamino acid residues in the FVII polypeptide. In some examples, a serumalbumin binding sequence replaces amino acid residues S103 to S111(corresponding to a mature FVII polypeptide set forth in SEQ ID NO:3) togenerate a modified FVII polypeptide. In other examples, a serum albuminbinding sequence replaces amino acid residues H115 to S126, or T128 toP134 (corresponding to a mature FVII polypeptide set forth in SEQ IDNO:3). Exemplary serum albumin binding sequences are set forth in SEQ IDNOS: 206-212.

Table 10 provides non-limiting examples of exemplary modifications thatcan be made to a FVII polypeptide insert a serum albumin bindingsequence. As noted above, inclusion of a serum albumin binding sequencecan increase the half-life of a FVII polypeptide. Thus, the modifiedFVII polypeptides can have increased coagulant activity. In reference tothe modifications listed in Table 10, the amino acid residues at whichthe serum albumin binding sequence is inserted in the FVII polypeptide,and the sequence of the binding sequence, are both represented in thetable. For example, S103S111delinsQRLMEDICLPRWGCLWEDDF indicates thatamino acid residues S103 through S111 of an unmodified FVII polypeptidefull length numbering (residues corresponding to the mature FVIIpolypeptide sequence set forth in SEQ ID NO:3) have been deleted andreplaced with a serum albumin binding sequence with the amino acidsequence QRLMEDICLPRWGCLWEDDF (SEQ ID NO:206). Recitation of just asingle amino acid residue, such as P406, indicates that the serumalbumin binding sequence is inserted after P406 and no amino acidresidues have been deleted from the FVII polypeptide. The amino acidpositions for mutation also are referred to by the chymotrypsinnumbering scheme where appropriate. In instances where a modified aminoacid position does not have a corresponding chymotrypsin number (i.e. isnot within amino acid positions 153 to 406 corresponding to a matureFVII polypeptide set forth in SEQ ID NO:3, and is not set forth in Table1, above), the position is denoted in brackets using mature FVIInumbering. For example, S103 does not have a corresponding chymotrypsinnumber and is set forth as S[103] when referring to chymotrypsinnumbering In Table 10 below, the sequence identifier (SEQ ID NO) isidentified in which exemplary amino acid sequences of the modified FVIIpolypeptide are set forth.

TABLE 10 Modification - mature FVII Modification - chymotrypsin SEQ IDnumbering numbering NO S103S111delinsQRLMEDICLPRWGS[103]S[111]delinsQRLMEDICLPRW 227 CLWEDDF GCLWEDDFH115S126delinsQRLMEDICLPRWG H[115]S[126]delinsQRLMEDICLPRW 228 CLWEDDFGCLWEDDF T128P134delinsQRLMEDICLPRWG T[128]P[134]delinsQRLMEDICLPRW 229CLWEDDF GCLWEDDF S103S111delinsIEDICLPRWGCLWES[103]S[111]delinsIEDICLPRWGCLWE 230 H115S126delinsIEDICLPRWGCLWEH[115]S[126]delinsIEDICLPRWGCL 231 WE T128P134delinsIEDICLPRWGCLWET[128]P[134]delinsIEDICLPRWGCLWE 232 S103S111delinsDICLPRWGCLWEDS[103]S[111]delinsDICLPRWGCLWED 233 H115S126delinsDICLPRWGCLWEDH[115]S[126]delinsDICLPRWGCLWED 234 T128P134delinsDICLPRWGCLWEDT[128]P[134]delinsDICLPRWGCLWED 235 P406insIEDICLPRWGCLWP257insIEDICLPRWGCLW 236 P406insGGGSIEDICLPRWGCLWP257insGGGSIEDICLPRWGCLW 237 P406insDICLPRWGCLWED P257insDICLPRWGCLWED238 P406insGGGSDICLPRWGCLWED P257insGGGSDICLPRWGCLWED 239

Modified FVII polypeptides containing a serum albumin binding sequencecan exhibit increased binding to serum albumin that is at least or about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to the bindingof unmodified or wild-type FVII polypeptide to serum albumin either invivo or in vitro. Modified FVII polypeptides that can bind to serumalbumin can exhibit increased serum half-life of at least about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 200%, 300%, 400%, 500%, or more compared to the serum half-life ofunmodified or wild-type FVII polypeptide either in vivo or in vitro.Increased serum albumin binding and/or increased serum half-life of suchmodified FVII polypeptides also can be manifested as increasedcoagulation activity, duration of coagulant activity and/or enhancedtherapeutic index. The coagulation activity of the modified FVIIpolypeptides can be increased by at least about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,300%, 400%, 500%, or more compared to the coagulation activity ofunmodified or wild-type FVII polypeptide either in vivo or in vitro.

b. Exemplary FVII Polypeptides with Platelet Integrin α_(IIb)β₃ BindingSequences

Provided herein are modified FVII polypeptides containing plateletintegrin α_(IIb)β₃ binding sequences. Platelet integrin α_(IIb)β₃ (alsocalled glycoprotein (GP) IIb/IIIa) is the most abundant plateletadhesion receptor. It is a calcium-dependent heterodimer that serves asa receptor for proteins including, but not limited to, fibrinogen,fibronectin, vitronectin, von Willebrand factor, and thrombospondin.Binding to “cognate” protein ligands can activate α_(IIb)β₃ and inducesignal transduction in the cytoplasm via the protein's intercellulardomain. Modified FVII polypeptides containing platelet integrinα_(IIb)β₃ binding sequences, therefore, can bind platelets. The modifiedFVII polypeptides can bind platelet integrin α_(IIb)β₃ (the activatedand/or unactivated form) in vitro or in vivo, resulting in an increasedhalf-life. Those FVIIa variants that bind selectively to activatedα_(IIb)β₃ can, therefore, be targeted to activated platelets and thusconcentrated at the site of an evolving blood clot. Selective targetingof FVIIa to evolving blood clots would be expected to improve thetherapeutic utility of the variant by improving both efficacy andtherapeutic index. Thus, provided herein are modified FVII polypeptideswith increased half-life compared to an unmodified FVII polypeptide andvariants that, in addition, bind selectively to activated platelets.When evaluated in an appropriate in vitro assay, or in vivo, such asfollowing administration to a subject as a pro-coagulant therapeutic,the modified FVII polypeptides can display increased coagulant activityas compared with unmodified FVII polypeptides.

The modified FVII polypeptides provided herein contain platelet integrinα_(IIb)β₃ binding sequences. Platelet integrin α_(IIb)β₃ bindingsequences can be inserted with the unmodified FVII polypeptide or can belinked to the C- or N-terminal of the FVII polypeptide. For example, theα_(IIb)β₃ binding sequences can extend from the proline residue at aminoacid position 406 at the C-terminus of the FVII polypeptide(corresponding to a mature FVII polypeptide set forth in SEQ ID NO:3).If the binding sequences are inserted within the FVII polypeptide,insertion is at a position such that the resulting modified FVIIpolypeptide retains at least one activity of an unmodified FVIIpolypeptide. The binding sequence can be inserted into the FVIIpolypeptide without removing any amino acid residues in the FVIIpolypeptide, or can replace one or more amino acid residues in the FVIIpolypeptide. In some examples, a platelet integrin α_(IIb)β₃ bindingsequence replaces amino acid residues S103 to S111 (corresponding to amature FVII polypeptide set forth in SEQ ID NO:3) to generate a modifiedFVII polypeptide. In other examples, an α_(IIb)β3 binding sequencereplaces amino acid residues H115 to S126, or T128 to P134(corresponding to a mature FVII polypeptide set forth in SEQ ID NO:3).Exemplary platelet integrin α_(IIb)β₃ binding sequences are set forth inSEQ ID NOS: 213-215.

Table 11 provides non-limiting examples of exemplary modifications thatcan be made to a FVII polypeptide to insert a platelet integrinα_(IIb)β₃ binding sequence. As noted above, inclusion of a plateletintegrin α_(IIb)β₃ binding sequence can increase the serum half-life ofa FVII polypeptide and/or target the protein to an evolving the bloodclot. Thus, the modified FVII polypeptides can have increased coagulantactivity. In reference to the modifications listed in Table 11, theamino acid residues at which the platelet integrin α_(IIb)β₃ bindingsequence is inserted in the FVII polypeptide, and the sequence of thebinding sequence, are both represented in the table. For example,H115S126delinsSFGRGDIRNV indicates that amino acid residues H115 thruS126 of an unmodified FVII polypeptide full length numbering (residuescorresponding to the mature FVII polypeptide sequence set forth in SEQID NO: 3) have been deleted, and replaced with an α_(IIb)β₃ bindingsequence with the amino acid sequence SFGRGDIRNV (SEQ ID NO:213).Recitation of just a single amino acid residue, such as P406, indicatesthat the α_(IIb)β₃ binding sequence is inserted after P406 and no aminoacid residues have been deleted from the FVII polypeptide. The aminoacid positions for mutation also are referred to by the chymotrypsinnumbering scheme where appropriate. In instances where a modified aminoacid position does not have a corresponding chymotrypsin number (i.e. isnot within amino acid positions 153 to 406 corresponding to a matureFVII polypeptide set forth in SEQ ID NO:3, and is not set forth in Table1, above), the position is denoted in brackets using mature FVIInumbering. For example, S103 does not have a corresponding chymotrypsinnumber and is set forth as S[103] when referring to chymotrypsinnumbering. In Table 11 below, the sequence identifier (SEQ ID NO) isidentified in which exemplary amino acid sequences of the modified FVIIpolypeptide are set forth.

TABLE 11 Modification - chymotrypsin Modification - mature FVIInumbering numbering SEQ ID NO S103S111delinsSFGRGDIRNVS[103]S[111]delinsSFGRGDIRNV 240 H115S126delinsSFGRGDIRNVH[115]S[126]delinsSFGRGDIRNV 241 T128P134delinsSFGRGDIRNVT[128]P[134]delinsSFGRGDIRNV 242 P406insCSFGRGDIRNVC P257insCSFGRGDIRNVC243 P406insGGGSCSFGRGDIRNVC P257insGGGSCSFGRGDIRNVC 244

Modified FVII polypeptides containing a platelet integrin α_(IIb)β₃binding sequence can exhibit increased binding to platelet integrinα_(IIb)β₃ that is at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%,500%, or more compared to the binding of unmodified or wild-type FVIIpolypeptide to platelet integrin α_(IIb)β₃ in vivo. Modified FVIIpolypeptides that can bind to platelets via platelet integrin α_(IIb)β₃can exhibit increased half-life of at least about 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,300%, 400%, 500%, or more compared to the half-life of unmodified orwild-type FVII polypeptide either in vitro, in vivo or ex vivo.Increased half-life of such modified FVII polypeptides also can bemanifested as increased coagulation activity, duration of coagulantactivity and/or enhanced therapeutic index. For example, the coagulationactivity of the modified FVII polypeptides can be increased by at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to thecoagulation activity of unmodified or wild-type FVII polypeptide eitherin vivo or in vitro.

6. Modification by Introduction of a Heterologous Gla Domain

Interaction of residues in the γ-carboxylated Gla domain of vitaminK-dependent plasma proteins, such as FVII, FIX, FX, prothrombin, proteinC and protein S, and negatively charged phospholipids on the membranesurface is important for hemostasis. The Gla domains of vitaminK-dependent plasma proteins typically contain approximately 45 aminoacids, of which 9 to 12 glutamic acid residues are post-translationallymodified by vitamin K-dependent carboxylation to form γ-carboxyglutamate(Gla). The amino acids that form the Gla domain are positionedimmediately after those that form the signal peptide and propeptide ofthe proteins, and are therefore situated at the N-terminus followingprocessing and cleavage of the precursor polypeptides to the matureproteins. For example, the amino acids that form the Gla domain in FVIIare at positions 39-83 of the precursor polypeptide set forth in SEQ IDNO:1, positions 61-105 of the precursor polypeptide set forth in SEQ IDNO:2, and positions 1 to 45 of the mature polypeptide set forth in SEQID NO:3. Of these, the 10 glutamic acid residues at positions E6, E7,E14, E19, E20, E25, E26, E29 and E35 of the mature FVII polypeptide setforth in SEQ ID NO: 3 are modified by carboxylation to generateγ-carboxyglutamate (Gla) residues.

Due to its relatively low binding affinity for activated platelets, theGla domain of FVII is a target for modification, with the aim ofenhancing the interaction between the modified FVII and the phospholipidmembrane, thereby increasing coagulation activity. Modification can beeffected by substitution of specific amino acids that are involved inthis interaction (see, e.g., Shah et al. PNAS 95: 4429-4234, Harvey etal. (2003) J Biol Chem 278:8363-8369). Alternatively, modification canbe effected by substitution of the entire Gla domain with the Gla domainof another vitamin K-dependent protein i.e. Gla domain swap. This typeof modification results in a chimeric protein, such as that whichresulted when the Gla domain of protein C was replaced with the Gladomain of FVII (Geng et al. (1997) Thromb Haemost 77:926-933).

Typically, such modification includes introduction, such as by additionor substitution, of a heterologous Gla domain, or a sufficient portionthereof to effect phospholipids binding into a region of the FVIIpolypeptide to generate a chimeric modified FVII polypeptide. Generally,such a chimeric FVII polypeptide retains at least one activity of FVII.The binding and/or affinity of Gla-modified FVII polypeptides foractivated platelets can be increased by at least about 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,200%, 300%, 400%, 500%, or more compared to the binding and/or affinityof unmodified or wild-type FVII polypeptide either in vivo or in vitro.The binding and/or affinity for activated platelets by modified FVIIpolypeptides also can be manifested as increased coagulation activity.The coagulation activity of the Gla-modified FVII polypeptides can beincreased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, ormore compared to the coagulation activity of unmodified or wild-typeFVII polypeptide either in vivo or in vitro.

A Gla domain or sufficient portion thereof to effect phospholipidbinding, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more of the heterologous Gla domain,contained within any polypeptide can be used as a source of aheterologous Gla domain for introduction or replacement of a region of aFVII polypeptide. Typically, such a heterologous Gla domain exhibitsbinding affinity for phospholipids, for example, phospholipids presenton the surface of an activated platelet. Generally, the choice of aheterologous Gla domain is one that exhibits higher affinity forphospholipids as compared to the affinity of the Gla domain of FVII. Theexact Gla domain, or sufficient portion thereof, used as a heterologousdomain for modification of a FVII polypeptide can be rationally orempirically determined. Exemplary of other Gla-containing polypeptidesinclude, but are not limited to, FIX, FX, prothrombin, protein C,protein S, osteocalcin, matrix Gla protein, Growth-arrest-specificprotein 6 (Gas6), and protein Z. The Gla domains of these exemplaryproteins are set forth in any of SEQ ID NOS: 83-91. For example, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or more contiguous amino acids, or theentire Gla domain, of a heterologous Gla domain can be introduced into aFVII polypeptide. In addition, introduction of the Gla domain into aFVII polypeptide also can include additional amino acids not part of theGla domain of the heterologous polypeptide so long as the additionalamino acids do not significantly weaken the phospholipid binding abilityof the introduced Gla domain.

In some examples, the introduction is by addition of the Gla domain tothe FVII polypeptide such that the heterologous Gla domain is insertedinto the endogenous Gla domain or into another region or domain of theFVII polypeptide so long as the modified FVII polypeptide retains atleast one activity of FVII. In such examples, the native Gla domain ofthe FVII polypeptide is retained in the polypeptide, although in someinstances the amino acid sequence that makes up the native Gla domain isinterrupted. In other examples, the heterologous Gla domain, or asufficient portion thereof, is inserted adjacent to, either on the N- orC-terminus, of the native Gla domain such that the native Gla domain isnot interrupted. In an additional example, the heterologous Gla domain,or a sufficient portion thereof, is inserted into another domain of theFVII polypeptide.

Also provided herein are modified Gla-domain FVII polypeptides where allor a contiguous portion of the endogenous Gla domain of FVII is removedand is replaced with a heterologous Gla domain, or a sufficient portionthereof to effect phospholipid binding, so long as the modified FVIIpolypeptide retains at least one activity of FVII. Such modificationalso is referred to as a Gla domain swap. Exemplary of Gla swapmodifications are those in which the endogenous Gla domain is replacedwith all or a portion of the Gla domain of any one of FIX (SEQ IDNO:83), FX (SEQ ID NO:84), thrombin (SEQ ID NO:85), Protein C (SEQ IDNO:86) or Protein S (SEQ ID NO:87). Such modifications are called “GlaSwap FIX,” “Gla Swap FX,” “Gla Swap Thrombin,” “Gla Swap Prot C” and“Gla Swap Prot S,” respectively. Such modified FVII polypeptides canexhibit increased binding to activated platelets, resulting in increasedcoagulant activity. The “Gla swap FIX” modification involves deletion ofthe endogenous FVII Gla domain by deleting amino acid residues A1 to Y44(residues corresponding to a mature FVII polypeptide set forth in SEQ IDNO:3) and insertion of 45 amino acid residues that correspond to aminoacid residues Y1 to Y45 of the FIX Gla domain set forth in SEQ ID NO:83.The Gla Swap FX modification involves deletion of amino acid residues A1to Y44 (residues corresponding to a mature FVII polypeptide set forth inSEQ ID NO:3) and insertion of 44 amino acid residues that correspond toA1 to Y44 of the FX Gla domain set forth in SEQ ID NO:84. The Gla SwapThrombin modification involves deletion of amino acid residues A1 to Y44(residues corresponding to a mature FVII polypeptide set forth in SEQ IDNO:3) and insertion of 44 amino acid residues that correspond to aminoacid residues Y1 to Y44 of the Thrombin Gla domain set forth in SEQ IDNO:85. The Gla Swap Protein C modification involves deletion of aminoacid residues A1 to Y44 (residues corresponding to a mature FVIIpolypeptide set forth in SEQ ID NO:3) and insertion of 44 amino acidresidues that correspond to amino acid residues A1 to H44 of the ProteinC Gla domain set forth in SEQ ID NO:86. The Gla Swap Protein Smodification involves deletion of amino acid residues A1 to Y44(residues corresponding to a mature FVII polypeptide set forth in SEQ IDNO:3) and insertion of 44 amino acid residues that correspond to aminoacid residues Y1 to Y44 of the Protein S Gla domain set forth in SEQ IDNO:87.

In some examples, modifications, including, but not limited to, aminoacid substitutions or replacements, insertions and/or deletions, aremade to the heterologous Gla domain that is being introduced into theFVII polypeptide. Such modifications can effect, for example, increasedbinding to activated platelets, due to increased phospholipid binding,as compared to the binding observed with the wild type form of theheterologous Gla domain. For example, if the Factor IX Gla domain, or aphospholipid binding portion thereof, is introduced into a FVIIpolypeptide to generate a modified FVII polypeptide, the Factor IX Gladomain can contain amino acid mutations that confer increasedphospholipid binding compared to the wild-type Factor IX Gla domain. Theheterologous Gla domain contained the modified FVII polypeptidesprovided herein can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moremodifications, such as amino acid substitutions or replacements,insertions and/or deletions.

In some examples, the modification(s) in the heterologous Gla domainincrease phospholipid binding. In other examples, the heterologous Gladomain can contain one or more mutations compared to the wild-type formof the heterologous Gla domain that confer FVII-like functions to theheterologous Gla domain. For example, as noted above, R36 of the FVIIGla domain set forth in SEQ ID NO:119 can be involved in interactionswith FX. Hence, the heterologous Gla domain can contain furthermodifications, such as any required to maintain an arginine at position36 of the mature FVII polypeptide, as set forth in SEQ ID NO:3, or anyother modifications required to maintain FX-activation properties of themodified FVIIa polypeptide (Ruf et al. (1999) Biochem 38:1957-1966).Thus, in some examples, a corresponding mutation to R36 can be made inthe heterologous Gla domain. The corresponding position can bedetermined by one of skill in the art, such as by alignment of aminoacid sequences.

Provided herein are modified FVII polypeptides containing a Gla swapmodification wherein the heterologous Gla domain, or phospholipidbinding portion thereof, contains one or more mutations compared to thewild-type heterologous Gla domain, and is introduced into the FVIIpolypeptide by replacement of some or all of the endogenous FVII Gladomain. In one example, the modified FVII polypeptides provided hereincontain a “Gla swap FIX” modification, which, as described above,involves deletion of the endogenous FVII Gla domain by deleting aminoacid residues A1 to Y44 (residues corresponding to a mature FVIIpolypeptide set forth in SEQ ID NO:3) and insertion of 45 amino acidresidues that correspond to amino acid residues Y1 to Y45 of the FIX Gladomain set forth in SEQ ID NO:83. The FIX Gla domain used in the Glaswap modification can contain one or more mutations compared to the wildtype form of the FIX Gla domain set forth in SEQ ID NO:83, such as 1, 2,3, 4, 5 or more mutations, such as amino acid substitutions, deletionsor insertions. For example, the heterologous FIX Gla domain in the “Glaswap FIX” modified FVII polypeptide can contain one or more amino acidsubstitutions at amino acid positions corresponding to M19, E40, K43and/or Q44 of the FIX Gla domain set forth in SEQ ID NO:83.

In one example, the FIX Gla domain contains a M19K amino acidsubstitution. Such a modification is denoted by {Gla Swap FIX/M19K} i.e.the methionine at the amino acid position corresponding to amino acidposition 19 of the FIX Gla domain set forth in SEQ ID NO:83 is replacedwith a lysine. In a further example, the modified heterologous FIX Gladomain in the modified FVII polypeptide contains a E40L amino acidsubstitution, denoted by {FIX Gla Swap/E40L}, whereby the glutamic acidat the amino acid position corresponding to amino acid position 40 ofthe FIX Gla domain set forth in SEQ ID NO:83 is replaced with a leucine.Also provided herein are modified FVII polypeptides that contain a K43Isubstitution (denoted by {Gla Swap FIX/K43I}) wherein the lysine at theamino acid position corresponding to amino acid position 43 of the FIXGla domain set forth in SEQ ID NO:83 is replaced with an isoleucine. Inanother example, the modified heterologous FIX Gla domain in themodified FVII polypeptide contains a Q44S amino acid substitution,denoted by {FIX Gla Swap/Q44S}, whereby the glutamine at the amino acidposition corresponding to amino acid position 44 of the FIX Gla domainset forth in SEQ ID NO:83 is replaced with a serine. In one example, theheterologous FIX Gla domain contains the M19K/E40L/K43I/Q44S amino acidsubstitutions.

Modified FVII polypeptides containing a heterologous Gla domain, such asmodified heterologous Gla domain, can exhibit increased coagulantactivity at lower dosages as compared to a wild-type FVII molecule, suchas NovoSeven®, due to increased binding and/or affinity for activatedplatelets. The coagulation activity of the Gla-modified FVIIpolypeptides can be increased by at least or about 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,300%, 400%, 500%, or more compared to the coagulation activity ofunmodified or wild-type FVII polypeptide either in vivo, ex vivo or invitro.

7. Combinations and Additional Modifications

Any one or more of the modifications described above can be combinedwith any other modification(s) described above or described elsewhere inthe art. Thus, in addition to modification of FVII polypeptides to haveincreased resistance to AT-III, increased catalytic activity, increasedresistance to inhibition by Zn²⁺, altered glycosylation, improvedpharmacokinetic properties, such as increased half-life, increasedbinding and/or affinity to serum albumin, increased binding and/oraffinity to phospholipids, or increased binding and/or affinity forplatelet integrin platelet integrin α_(IIb)β₃, modified FVIIpolypeptides provided herein also include those that exhibit more thanone of the above-noted properties. Typically, such additionalmodifications are those that themselves result in an increased coagulantactivity of the modified polypeptide and/or increased stability of thepolypeptide. Accordingly, the resulting modified FVII polypeptidesexhibit an increased coagulant activity. The additional modificationscan include, for example, any amino acid substitution, deletion orinsertion known in the art, typically any that increases the coagulantactivity and/or stability of the FVII polypeptide. Any modified FVIIpolypeptide provided herein can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional amino acidmodifications, so long as the resulting modified FVII polypeptideretains a FVII activity of the wild-type or unmodified polypeptide.

In one example, the additional modification can be made to the FVIIpolypeptide sequence such that its interaction with other factors,molecules and proteins is altered. For example, the amino acid residuesthat are involved in the interaction with tissue factor pathwayinhibitor (TFPI) can be replaced such that the affinity and/or bindingof the modified FVII polypeptide to TF is decreased. Other modificationsinclude, but are not limited to, modification of amino acids that areinvolved in interactions with factor X, factor IX, tissue factor (TF)and phospholipids. In some examples, the modification made to the FVIIpolypeptide sequence includes insertion of amino acids that constitute abinding sequence, such as, for example, a serum albumin binding sequenceor a glycoprotein IIb-IIIa binding sequence.

Additional modifications also can be made to a modified FVII polypeptideprovided herein that alter the conformation or folding of thepolypeptide. These include, for example, the replacement of one or moreamino acids with a cysteine such that a new disulphide bond is formed,or modifications that stabilize an α-helix conformation, therebyimparting increased activity to the modified FVII polypeptide.

Additional modifications also can be made to the FVII polypeptide toeffect post-translational modifications. For example, the polypeptidecan be modified to include additional glycosylation sites such that theresulting modified FVII polypeptide has increased glycosylation comparedto an unmodified FVII polypeptide. Modifications also can be made tointroduce amino acid residues that can be subsequently linked to achemical moiety, such as one that acts to increase stability of themodified FVII polypeptide. The stability of a FVII polypeptide also canbe altered by modifying potential proteolytic sites, thereby increasingthe resistance of the modified FVII polypeptide to proteases.

Additionally, amino acids substitutions, deletions or insertions can bemade in the endogenous Gla domain such that the modified FVIIpolypeptide displays increased binding and/or affinity for phospholipidmembranes. Such modifications can include single amino acidsubstitution, deletions and/or insertions, or can include amino acidsubstitution, deletion or insertion of multiple amino acids. Forexample, all or part of the endogenous Gla domain can be replaced withall or part of a heterologous Gla domain. In other examples, themodified FVII polypeptides provided herein can display deletions in theendogenous Gla domain, or substitutions in the positions that arenormally gamma-carboxylated (US20070037746).

The following sections describe non-limiting examples of exemplarymodifications described in the art to effect increased stability and/orcoagulant activity of a FVII polypeptide. As discussed above, suchmodifications also can be additionally included in any modified FVIIpolypeptide provided herein. The amino acid positions referenced belowcorrespond to the mature FVII polypeptide as set forth in SEQ ID NO:3.Corresponding mutations can be made in other FVII polypeptides, such asallelic, species or splices variants of the mature FVII polypeptide setforth in SEQ ID NO:3.

a. Modifications that Increase Resistance to TFPI

In one example, additional modifications can be made to a modified FVIIpolypeptide that contains a modification at amino acid position 286 bymature FVII numbering that result in increased resistance to TFPI. Suchresistance to TFPI can be achieved, for example, by mutation of one ormore residues in FVII involved in the interaction and binding with TFPIto reduce or prevent such binding, thereby making the modified FVIIpolypeptides resistant to the naturally inhibitory effects of TFPI withrespect to coagulation initiation. For example, the modifications can bemade at amino acid residues that are FVII/TFPI contact residues orresidues in close proximity to the interaction surface.

Examples of additional modifications that can be included in themodified FVII polypeptides provided herein to increase resistance toTFPI include, but are not limited to, those described in InternationalPatent Publication No. WO2004/083361, Neuenschwander et al., (1995)Biochemistry 34:8701-8707, Chang et al., (1999) Biochemistry38:10940-10948, and Iakhiaev et al., (2001) Thromb. Haemost. 85:458-463,and related application U.S. application Ser. No. 12/082,662.Non-limiting examples of exemplary amino acid modifications described inthe art that can result in increased resistance to TFPI of the modifiedFVII polypeptide include any one or more of Q176, D196K, D196R, D196A,D196Y, D196F, D196W, D196L, D196I, K197Y, K197A, K197E, K197D, K197L,K197M, K197I, K197V, K197F, K197W, K199A, K199D, K199E, G237W, G237T,G237I, G237V, T239A, R290A, R290E, R290D, R290N, R290Q, R290K, R290M,R290V, K341E, K341R, K341Q, K341N, K341M, K341D, G237T238insA,G237T238insS, G237T238insV, G237T238insAS, G237T238insSA, D196K197insK,D196K197insR, D196K197insY, D196K197insW, D196K197insA, D196K197insM,K197I198insE, K197I198insY, K197I198insA and K197I198insS (where, forexample, G237T238insAS denotes a modification in which an alanine (A)and a serine (S) have been inserted between the glycine at position 237(G237) and the threonine at position 238 (T238).

b. Modifications that Increase Intrinsic Activity

In one example, additional modifications can be made to a modifiedfactor VII polypeptide provided herein that result in increasedcatalytic activity toward factor X. For example, modifications can bemade to the amino acids that are involved in the interaction with itscofactor, TF, such that the resulting modified FVII polypeptide hasincreased affinity for TF, and thereby displays increased activitytoward FX. Modifications also can be made to the activation pocket ofthe FVII polypeptide, such that the intrinsic activity of the modifiedFVII polypeptide toward FX is increased compared to the activity of theunmodified polypeptide. Another modification strategy that results inincreased activity involves modification of the FVII polypeptide suchthat the folding and conformation of the protein is altered to a moreactive form. For example, amino acid substitutions can be made such thatthe α-helix loop region (corresponding to positions 305 to 321 of themature sequence as set forth in SEQ ID NO:3) of the protease domain isstabilized and folded more tightly to the body of the protease domain toconfer a more zymogen-like shape on the modified FVII polypeptide. Amore active polypeptide also can be achieved by modification of theamino acids involved in the β-strands of the FVII polypeptide. Forexample, amino acid substitutions can be made that introduce newcysteine pairs that can form new disulphide bonds which can function to“lock” the modified FVII polypeptide into a more active form.

Examples of additional modifications that can be included in themodified FVII polypeptides provided herein to increase the intrinsicactivity of the modified FVII polypeptide include, but are not limitedto, those described in Persson et al. (2004) Biochem J. 379:497-503,Maun et al. (2005) Prot Sci 14:1171-1180, Persson et al. (2001) PNAS98:13583-13588, Persson et al. (2002) Eur J Biochem 269:5950-5955,Soejima et al. (2001) J Biol Chem 276:17229-17235, Soejima et al. (2002)J Biol Chem 277:49027-49035, WO200183725, WO2002022776, WO2002038162,WO2003027147, WO200338162, WO2004029090, WO2004029091, WO2004108763 andWO2004111242. Non-limiting examples of exemplary amino acidmodifications described in the art that can result in increasedintrinsic activity of the modified FVII polypeptide include any one ormore of S279C/V302C, L280C/N301C, V281C/V302C, S282C/V299C, S314E, L39E,L39Q, L39H, I42R, S43Q, S53N, K62E, K62R, K62D, K62N, K62Q, K62T, L65Q,L65S, F71D, F71Y, F71E, F71Q, F71N, P74S, P74A, A75E, A75D, E77A, E82Q,E82N, T83K, E116D, K157V, K157L, K1571, K157M, K157F, K157W, K157P,K157G, K157S, K157T, K157C, K157Y, K157N, K157E, K157R, K157H, K157D,K157Q, V158L, V158I, V158M, V158F, V158W, V158P, V158G, V158S, V158T,V158C, V158Y, V158N, V158E, V158R, V158K, V158H, V158D, V158Q, A274M,A274L, A274K, A274R, A274D, A274V, A2741, A274F, A274W, A274P, A274G,A274T, A274C, A274Y, A274N, A274E, A274H, A274S, A274Q, F275H, E296V,E296L, E2961, E296M, E296F, E296W, E296P, E296G, E296S, E296T, E296C,E296Y, E296N, E296K, E296R, E296H, E296D, E296Q, M298Q, M298V, M298L,M298I, M298F, M298W, M298P, M298G, M298S, M298T, M298C, M298Y, M298N,M298K, M298R, M298H, M298E, M298D, R304Y, R304F, R304L, R304M, L305V,L305Y, L305I, L305F, L305A, L305M, L305W, L305P, L305G, L305S, L305T,L305C, L305N, L305E, L305K, L305R, L305H, L305D, L305Q, M306D, M306N,D309S, D309T, S314A, S314V, S3141, S314M, S314F, S314W, S314P, S314G,S314L, S314T, S314C, S314Y, S314N, S314E, S314K, S314R, S314H, S314D,S314Q, D334G, D334E, D334A, D334V, D334I, D334M, D334F, D334W, D334P,D334L, D334T, D334C, D334Y, D334N, D334K, D334R, D334H, D334S, D334Q,S336G, S336E, S336A, S336V, S3361, S336M, S336F, S336W, S336P, S336L,S336T, S336C, S336Y, S336N, S336K, S336R, S336H, S336D, S336Q, K337L,K337V, K337I, K337M, K337F, K337W, K337P, K337G, K337S, K337T, K337C,K337Y, K337N, K337E, K337R, K337H, K337D, K337Q, F374P, F374A, F374V,F3741, F374L, F374M, F374W, F374G, F374S, F374T, F374C, F374Y, F374N,F374E, F374K, F374R, F374H, F374D, F374Q, and substitution of positions300-322, 305-322, 300-312, or 305-312 with the corresponding amino acidsfrom trypsin, thrombin or FX, and substitution of positions 310-329,311-322 or 233-329 with the corresponding amino acids from trypsin.

c. Modifications that Increase Resistance to Proteases

Modified FVII polypeptides provided herein also can contain additionalmodifications that result in increased resistance of the polypeptide toproteases. For example, amino acid substitutions can be made that removeone or more potential proteolytic cleavage sites. The modified FVIIpolypeptides can thus be made more resistant to proteases, therebyincreasing the stability and half-life of the modified polypeptide.

Examples of additional modifications that can be included in themodified FVII polypeptides provided herein to increase resistance toproteases include, but are not limited to, those described in U.S. Pat.No. 5,580,560 or International Published Application Nos. WO1988010295and WO2002038162. Non-limiting examples of exemplary modificationsdescribed in the art that can result in increased resistance of themodified FVII polypeptide to inhibitors and/or proteases include any oneor more of K32Q, K32E, K32G, K32H, K32T, K32A, K32S, K38T, K38D, K38L,K38G, K38A, K38S, K38N, K38H, I42N, I42S, I42A, I42Q, Y44N, Y44S, Y44A,Y44Q, F278S, F278A, F278N, F278Q, F278G, R290G, R290A, R290S, R290T,R290K, R304G, R304T, R304A, R304S, R304N, R315G, R315A, R315S, R315T,R315Q, Y332S, Y332A, Y332N, Y332Q, Y332G, K341E, K341Q, K341G, K341T,K341A and K341S.

d. Modifications that Increase Affinity for Phospholipids

The modified FVII polypeptide provided herein also can contain one ormore additional modifications to increase affinity for phospholipids.The coagulant activity of FVII can be enhanced by increasing the bindingand/or affinity of the polypeptide for phospholipids, such as thoseexpressed on the surface of activated platelets. This can be achieved,for example, by modifying the endogenous FVII Gla domain. Modificationcan be effected by amino acid substitution at one or more positions inthe Gla domain of a FVII polypeptide that result in a modified FVIIpolypeptide with increased ability to bind phosphatidylserine and othernegatively charged phospholipids. Examples of additional modificationsto increase phospholipid binding and/or affinity and that can be made toa modified FVII polypeptide provided herein that contains an endogenousFVII Gla domain, include, but are not limited to, those described inHarvey et al. (2003) J Biol Chem 278:8363-8369, US20030100506,US20040220106, US20060240526, US6017882, U.S. Pat. No. 6,693,075, U.S.Pat. No. 6,762,286, WO200393465 and WO2004111242. Exemplary of suchmodifications include any one or more of: an insertion of a tyrosine atposition 4, or modification of any one or more of P10Q, P10E, P10D,P10N, R28F, R28E, K32E, K32D, D33F, D33E, D33K A34E, A34D, A34I, A34L,A34M, A34V, A34F, A34W, A34Y, R36D, R36E, K38E and K38D.

e. Modifications that Alter Glycosylation

Alteration of the extent, level and/or type of glycosylation of aprotein has been described in the art as a means to reduceimmunogenicity, increase stability, reduce the frequency ofadministration and/or reduce adverse side effects such as inflammation.Normally, this is effected by increasing the glycosylation levels. Theglycosylation site(s) provides a site for attachment for a carbohydratemoiety on the polypeptide, such that when the polypeptide is produced ina eukaryotic cell capable of glycosylation, it is glycosylated.

There are four native glycosylation sites in FVII; two N-glycosylationsites at N145 and N322, and two O-glycosylation sites at S52 and S60,corresponding to amino acid positions in the mature FVII polypeptide setforth in SEQ ID NO:3. In one embodiment, additional modifications can bemade to a modified FVII polypeptide provided herein such thatglycosylation at the above sites is disrupted. This can result in amodified FVII polypeptide with increased coagulant activity (see, e.g.,WO2005123916). Non-limiting examples of exemplary modificationsdescribed in the art that can result in decreased glycosylation andincreased activity of the modified FVII polypeptide as compared to anunmodified FVII polypeptide include, but are not limited to S52A, S60A,N145Y, N145G, N145F, N145M, N145S, N145I, N145L, N145T, N145V, N145P,N145K, N145H, N145Q, N145E, N145R, N145W, N145D, N145C, N322Y, N322G,N322F, N322M, N322S, N322I, N322L, N322T, N322V, N322P, N322K, N322H,N322Q, N322E, N322R, N322W and N322C.

In another embodiment, further modifications can be made to the aminoacid sequence of the modified FVII polypeptides provided herein suchthat additional glycosylation sites are introduced, thus increasing thelevel of glycosylation of the modified FVII polypeptide as compared toan unmodified FVII polypeptide. The glycosylation site can be anN-linked or O-linked glycosylation site. Examples of modifications thatcan be made to a FVII polypeptide that introduce one or more newglycosylation sites include, but are not limited to, those that aredescribed in U.S. Pat. No. 6,806,063 and WO200393465. Non-limitingexamples of exemplary modifications described in the art that can resultin increased glycosylation of the modified FVII polypeptide as comparedto an unmodified FVII polypeptide include, but are not limited to F4S,F4T, P10N, Q21N, W41N, S43N, A51N, G58N, L65N, G59S, G59T, E82S, E82T,N95S, N95T, G97S, G97T, Y101N, D104N, T106N, K109N, G117N, G124N, S126N,T128N, A175S, A175T, G179N, 1186S, 1186T, V188N, R202S, R202T, I205S,I205T, D212N, E220N, 1230N, P231N, P236N, G237N, V253N, E265N, T267N,E270N, R277N, L280N, G291N, P303S, P303ST, L305N, Q312N, G318N, G331N,D334N, K337N, G342N, H348N, R353N, Y357N, 1361N, V376N, R379N, M391N,K32N/A34S, K32N/A34T, F31N/D33S, F31N/D33T, I30N/K32S, I30N/K32T,A34N/R36S, A34N/R36T, K38N/F40S, K38N/F40T, T37N/L39S, T37N/L39T,R36N/K38S, R36N/K38T, L39N/W41 S, L39N/W41T, F40N/142S, F40N/142T,I42N/Y44S, I42N/Y44T, Y44N/D46S, Y44N/D46T, D46N/D48S, D46N/D48T,G47N/Q49S, G47N/Q49T, S52N/P54S, Q66N/Y68S, S119N/L121S, A122N/G124S,T128N/P129A, T130N/E132S, K143N/N145S, K143N/N145T, E142N/R144S,E142N/R144T, L141N/K143S, L141N/K143T, I140N/E142S/, I140N/E142T,R144N/A146S, R144N/A146T, A146N/K148S, A146N/K148T, S147N/P149S/,S147N/P149T, R290N/A292S, R290N/A292T, A292N/A294S, D289N/G291S,D289N/G291T, L288N/R290S, L288N/R290T, L287N/D289S, L287N/D289T,A292N/A294S, A292N/A294T, T293N/L295S, T293N/L295T, R315N/N317S,R315N/N317T, S314N/K316S, S314N/K316T, Q313N/R315S, Q313N/R315T,K316N/G318S, K316N/G318T, V317N/D319S, V317N/D319T, K341N/D343S,K341N/D343T, S339N/K341S, S339N/K341T, D343N/G345S, D343N/G345T,R392N/E394S, R392N/E394T, L390N/R392S, L390N/R392T, K389N/M391S,K389N/M391T, S393N/P395S, S393N/P395T, E394N/R396S, E394N/R396T,E394N/P395A/R396S, P395N/P397S, P395N/P397T, R396N/G398S, R396N/G398T,P397N/V399S, P397N/V399T, G398N/L400S, G398N/L400T, V399N/L401S,V399N/L401T, L400N/R402S, L400N/R402T, L401N/A4035, L401N/A403T,R402N/P404S, R402N/P404T, A403N/F405S, A403N/F405T, P404N/P406S andP404N/P406T.

f. Modifications to Facilitate Chemical Group Linkage

Additional modifications of a modified FVII polypeptide provided hereinalso can be made to facilitate subsequent linkage of a chemical group.One or more amino acid substitutions or insertions can be made such thata chemical group can be linked to a modified FVII polypeptide via thesubstituted amino acid. For example, a cysteine can be introduced to amodified FVII polypeptide, to which a polyethylene glycol (PEG) moietycan be linked to confer increased stability and serum half-life. Otherattachment residues include lysine, aspartic acid and glutamic acidresidues. In some embodiments, amino acids residues are replaced toreduce the number of potential linkage positions. For example, thenumber of lysines can be reduced. Examples of modifications that can bemade to the amino acid sequence of a FVII polypeptide which canfacilitate subsequent linkage with a chemical group include, but are notlimited to, those that are described in US20030096338, US20060019336,U.S. Pat. No. 6,806,063, WO200158935 and WO2002077218. Non-limitingexamples of exemplary modifications of a FVII polypeptides that canfacilitate subsequent linkage with a chemical group include, but are notlimited to Q250C, R396c, P406C, I42K, Y44K, L288K, D289K, R290K, G291K,A292K, T293K, Q313K, S314K, R315K, V317K, L390K, M391K, R392K, S393K,E394K, P395K, R396K, P397K, G398K, V399K, L400K, L401K, R402K, A403K,P404K, F405K, I30C, K32C, D33C, A34C, T37C, K38C, W41C, Y44C, S45C,D46C, L141C, E142C, K143C, R144c, L288C, D289C, R290c, G291C, A292C,S314C, R315c, K316C, V317C, L390C, M391C, R392C, S393C, E394C, P395C,R396c, P397C, G398C, V399C, L401C, R402c, A403C, P404C, I30D, K32D,A34D, T37D, K38D, W41D, Y44D, S45D, D46C, L141D, E142D, K143D, R144D,L288D, R290D, G291D, A292D, Q313D, S314D, R315D, K316D, V317D, L390D,M391D, R392D, S393D, P395D, R396D, P397D, G398D, V399D, L401D, R402D,A403D, P404D, 130E, K32E, A34E, T37E, K38E, W41E, Y44E, S45E, D46C,L141E, E142E, K143E, R144E, L288E, R290E, G291E, A292E, Q313E, S314E,R315E, K316E, V317E, L390E, M391E, R392E, S393E, P395E, R396E, P397E,G398E, V399E, L401E, R402E, A403E, P404E, K18R, K32R, K38R, K62R, K85R,K109R, K137R, K143R, K148R, K157R, K161R, K197R, K199R, K316R, K337R,K341R, K389R, K18Q, K32Q, K38Q, K62Q, K85Q, K109Q, K137Q, K143Q, K148Q,K157Q, K161Q, K197Q, K199Q, K316Q, K337Q, K341Q, K389Q, K18N, K32N,K38N, K62N, K85N, K109N, K137N, K143N, K148N, K157N, K161N, K197N,K199N, K316N, K337N, K341N, K389N, K18H, K32H, K38H, K62H, K85H, K109H,K137H, K143H, K148H, K157H, K161H, K197H, K199H, K316H, K337H, K341H andK389H.

g. Exemplary Combination Mutations

Provided herein are modified FVII polypeptides that have two or moremodifications designed to affect one or properties or activities of anunmodified FVII polypeptide. In some examples, the two or moremodifications alter two or more properties or activities of the FVIIpolypeptide. The modifications can be made to the FVII polypeptides suchthat one or more of catalytic activity, resistance to AT-III, resistanceto TFPI, resistance to inhibition by Zn²⁺, intrinsic activity,amidolytic activity, phospholipid binding and/or affinity,glycosylation, resistance to proteases, half-life and interaction withother factors or molecules, such as FX, FIX, serum albumin and plateletintegrin α_(IIb)β₃, is altered. Typically, the two or more modificationsare combined such that the resulting modified FVII polypeptide hasincreased coagulant activity, increased duration of coagulant activity,and/or an enhanced therapeutic index compared to an unmodified FVIIpolypeptide. The modifications can include amino acid substitution,insertion or deletion. The increased coagulant activity, increasedduration of coagulant activity, and/or an enhanced therapeutic index ofthe modified FVII polypeptide containing two or more modifications canbe increased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%,140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, or morecompared to the activity of the starting or unmodified FVIIapolypeptide.

Provided herein are modified FVII polypeptides that contain two or moremodifications that are introduced into an unmodified FVII polypeptide toalter two or more activities or properties. The modified FVIIpolypeptides can contain 2, 3, 4, 5, 6 or more modifications. Further,each modification can involve one or more amino acid residues. Forexample, a modified FVII polypeptide can contain two modifications eachof which is a single amino acid substitution. In another example, amodified FVII polypeptide can contain two modifications, one of which isa single amino acid substitution and the other of which involvesdeletion of more than one amino acid residue and then insertion of morethan one amino acid residue. For example, a modified FVII polypeptideprovided herein can contain the amino acid substitution S222A (residuescorresponding to a mature FVII polypeptide set forth in SEQ ID NO:3) todisrupt Zn²⁺ binding and a Gla Swap FIX modification, which involvesdeletion of the endogenous FVII Gla domain by deleting amino acidresidues A1 to Y44 (residues corresponding to a mature FVII polypeptideset forth in SEQ ID NO:3) and insertion of 45 amino acid residues thatcorrespond to amino acid residues Y1 to Y45 of the FIX Gla domain setforth in SEQ ID NO:83.

Modified FVII polypeptides provided herein can have two or moremodifications selected solely from those set forth in Tables 5 to 13. Inother examples, the modified FVII polypeptide contains two or moremodifications where one or more modifications are selected from thoseset forth in Tables 5 to 13 and one or more modifications are additionalmodifications that are not set forth in Tables 5 to 13, such as, forexample, modifications described in the art. In some examples, the oneor more additional modifications can be selected from those set forth inSection D.6.a-e, above. For example, a modified FVII polypeptide cancontain a modification at one or more of amino acid residues D196, K197,K199, G237, T239, R290 or K341 based on numbering of a mature FVII setforth in SEQ ID NO:3 (corresponding to D60, K60a, K60c, G97, T99, R147and K192, respectively, based on chymotrypsin numbering), which canincrease resistance to TFPI, and a modification at one or more aminoacid residues that affects intrinsic activity, such as, for example,V158 and M298, (V21 and M156, respectively, based on chymotrypsinnumbering). For example, a modified FVII polypeptide can contain twoamino acid substitutions that increase resistance to TFPI, such as K197Eand G237V, and one amino acid substitution that increases intrinsicactivity, such as M298Q, resulting in a FVII polypeptide with increasedcoagulant activity.

Exemplary of the combination modifications provided herein are thosethat include at least the Q286R mutation (numbering corresponding to themature FVII polypeptide set forth in SEQ ID NO:3; corresponding to Q143Rby chymotrypsin numbering). The modified FVII polypeptides containingthe Q286R modification can contain 1, 2, 3, 4, 5, 6 or more additionalmodifications. These additional modifications can be included to, forexample, alter catalytic activity, resistance to AT-III, resistance toTFPI, resistance to inhibition by Zn²⁺, intrinsic activity, amidolyticactivity, phospholipid binding and/or affinity, glycosylation,resistance to proteases, half-life and interaction with other factors ormolecules, such as FX, FIX, serum albumin and platelet integrinα_(IIb)β₃. Typically, the modified FVII polypeptides provided hereinthat contain two or more modifications, wherein one modification is theamino acid substitution Q286R, exhibit increased coagulant activitycompared to the wild-type FVII polypeptide.

In some examples, the modified FVII polypeptides containing two or moremodifications, wherein one is Q286R, exhibit increased catalytic andcoagulant activity compared to the wild type polypeptide as well ascompared to a FVII polypeptide containing any one of the mutationsalone. For example, provided herein are modified FVII polypeptides thatcontain both the Q286R and M289Q amino acid substitutions (Q286R/M298Qwith numbering corresponding to the mature FVII polypeptide set forth inSEQ ID NO:3; corresponding to Q143R/M156Q by chymotrypsin numbering).The Q286R/M298Q combination FVII mutant exhibits increased catalyticactivity for its substrate, Factor X, compared to wild type FVII, theQ286R single mutant and the M298Q single mutant (see e.g. Example 4,below). For example, in one study, the M298Q mutant exhibited acatalytic activity for FX, in the presence of TF, that was about 1.8 to2 times greater than that of the wild-type polypeptide, the catalyticactivity of the Q286R mutant was approximately 2.1 times greater thanthat of the wild-type FVII polypeptide, and the Q286R/M298Q mutantexhibited a catalytic activity for FX that was approximately 3.6-4.4times that of the catalytic activity of the wild-type polypeptide for FX(see Table 15, below).

Non-limiting exemplary combination modifications are provided in Table12. These exemplary combination modifications include two or moremodifications that are designed to alter two or more activities orproperties of a FVII polypeptide, including, but not limited to,resistance to TFPI, resistance to AT-III, intrinsic activity, amidolyticactivity, catalytic activity, Zn²⁺ binding, phospholipid binding and/oraffinity, glycosylation, resistance to proteases, half-life andinteraction with other factors or molecules, such as FX and FIX.Modified FVII polypeptides containing such combination modifications canhave increased coagulant activity, increased duration of coagulantactivity, and/or an enhanced therapeutic index. The modifications setforth in Table 12 below use the same nomenclature and numbering systemsas described in Tables 5 to 11, above. For example, the “Gla Swap FIX”modification involves deletion of the endogenous FVII Gla domain bydeleting amino acid residues A1 to Y44 (residues corresponding to amature FVII polypeptide set forth in SEQ ID NO:3) and insertion of 45amino acid residues that correspond to amino acid residues Y1 to Y45 ofthe FIX Gla domain set forth in SEQ ID NO:83, as described above. Insome examples, the “Gla Swap FIX” modification also contains one or moreamino acid substitutions in the FIX Gla domain portion compared to awild type FIX Gla domain, as discussed above. For example, the Gla SwapFIX modification also can include a M19K amino acid substitution(numbering corresponding amino acid positions of the FIX Gla domain setforth in SEQ ID NO:83). Such a modification is denoted by {Gla SwapFIX/M19K}, i.e. the modified FVII polypeptide contains a heterologousFIX Gla domain in which the methionine at the position corresponding toposition 19 of the FIX Gla domain set forth in SEQ ID NO:83 is replacedwith a lysine. Thus, modifications made to the heterologous FIX Gladomain portions are referenced using amino acid positions correspondingto amino acid positions of the mature wild type FIX polypeptide, or thewild type FIX Gla domain set forth in SEQ ID NO:83. Modifications madeto amino acid positions in the FVII polypeptide are referenced usingamino acid positions corresponding to amino acid positions of a matureFVII polypeptide as set forth in SEQ ID NO:3 and also are referred to bythe chymotrypsin numbering scheme. For example, a modified FVIIpolypeptide containing the Q286R modification (numbering correspondingto the mature FVII polypeptide set forth in SEQ ID NO:3), and a Gla swapFIX modification, wherein the FIX Gla domain contains the M19K aminoacid substitution (numbering corresponding amino acid positions of theFIX Gla domain set forth in SEQ ID NO:83), is denoted by {Gla SwapFIX/M19K}/Q286R. Similarly, the modification {Gla SwapFIX/Q44S}/Q286R/M298Q denotes that the FVII polypeptide contains a GlaSwap FIX modification wherein the glutamine at the amino acid positioncorresponding to amino acid position 44 of the FIX Gla domain set forthin SEQ ID NO:83 is replaced with a serine, and also contains the Q286Rand M298Q amino acid substitutions, with numbering corresponding to themature FVII polypeptide set forth in SEQ ID NO:3. In Table 12 below, thesequence identifier (SEQ ID NO) is identified in which exemplary aminoacid sequences of the modified FVII polypeptide are set forth.

TABLE 12 Modification - mature FVII Modification - chymotrypsin SEQ IDnumbering numbering NO Gla Swap FIX/Q286R Gla Swap FIX/Q143R 131Q286R/H257A H117A/Q143R 132 S222A/Q286R S82A/Q143R 133 Q286R/S222A/H257AS82A/H117A/Q143R 134 Gla Swap FIX/S222A/Q286R S82A/Gla Swap FIX/Q143R135 Gla Swap FIX/H257A/Q286R H117A/Gla Swap FIX/Q143R 136 Gla Swap FIX/Q143R/S82A/H117A/Gla Swap 137 S222A/H257A/Q286R FIX Q286R/M298QQ143R/M156Q 138 Q286R/M298Q/K341Q Q143R/M156Q/K192Q 139K199E/Q286R/M298Q K60cE/Q143R/M156Q 140 Gla Swap FIX/Q286R/M298Q GlaSwap FIX/Q143R/M156Q 141 Q286R/Q366V Q143R/Q217V 142Q286R/A292N/A294S/Q366V Q143R/A150N/A152S/Q217V 143 A175S/Q286R/Q366VA39S/Q143R/Q217V 144 S222A/Q286R/Q366V S82A/Q143R/Q217V 145 H257S/Q286RH117S/Q143R 146 H257S/Q286R/Q366V H117S/Q143R/Q217V 147S222A/H257A/Q286R/Q366V S82A/H117A/Q143R/Q217V 148 Q286R/H373AQ143R/H224A 149 S222A/H257A/Q286R/M298Q S82A/H117A/Q143R/M156Q 150Q286R/K341D Q143R/K192D 151 Q286R/Q366D Q143R/Q217D 152 Q286R/Q366NQ143R/Q217N 153 Q286R/M298Q/Q366D Q143R/M156Q/Q217D 154Q286R/M298Q/Q366N Q143R/M156Q/Q217N 155 Q286R/H373F Q143R/H224F 156Q286R/M298Q/H373F Q143R/M156Q/H224F 157 Gla Swap FIX/S222A Gla SwapFIX/S82A 245 Gla Swap FIX/H257A Gla Swap FIX/H117A 246 Gla SwapFIX/S222A/H257A Gla Swap FIX/S82A/H117A 247 S222A/M298Q S82A/M156Q 248H257A/M298Q H117A/M156Q 249 S222A/H257A/M298Q S82A/H117A/M156Q 250S222A/A292N/A294S/Q366V S82A/A150N/A152S/Q217V 251 A175S/S222A/Q366VA39S/S82A/Q217V 252 S222A/Q366V S82A/Q217V 253 H257S/Q366V H117S/Q217V254 S222A/H373A S82A/H224A 255 V158T/L287T/M298K V21T/L144T/M156K 256V158D/L287T/M298K V21D/L144T/M156K 257 S103S111delinsIEDICLPRWGCLWE/S[103]S[111]delinsIEDICLPRWG 258 G237V CLWE/G97VS103S111delinsDICLPRWGCLWED/ S[103]S[111]delinsDICLPRWGC 259 G237VLWED/G97V H115S126delinsQRLMEDICLPRWG H[115]S[126]delinsQRLMEDICL 260CLWEDDF/G237V PRWGCLWEDDF/G97V H115S126delinsIEDICLPRWGCLWE/H[115]S[126]delinsIEDICLPRWG 261 G237V CLWE/G97VH115S126delinsDICLPRWGCLWED/ H[115]S[126]delinsDICLPRWGC 262 G237VLWED/G97V T128P134delinsQRLMEDICLPRWG T[128]P[134]delinsQRLMEDICL 263CLWEDDF/G237V PRWGCLWEDDF/G97V T128P134delinsIEDICLPRWGCLWE/T[128]P[134]delinsIEDICLPRWG 264 G237V CLWE/G97VS103S111delinsQRLMEDICLPRWG S[103]S[111]delinsQRLMEDICL 265CLWEDDF/G237V PRWGCLWEDDF/G97V T128P134delinsDICLPRWGCLWED/T[128]P[134]DICLPRWGCLWED/ 266 G237V G97V S103S111delinsSFGRGDIRNV/G237VS[103]S[111]delinsSFGRGDIRNV/ 267 G97V H115S126delinsSFGRGDIRNV/G237VH[115]S[126]delinsSFGRGDIRNV/ 268 G97V T128P134delinsSFGRGDIRNV/G237VT[128]P[134]delinsSFGRGDIRNV/ 269 G97V M298Q/H373F M156Q/H224F 270S119N/L121S/A175S S[119]N/L[121]S/A39S 271 T128N/P129A/A175ST[128]N/P[129]A/A39S 272 A122N/G124S/A175S A[122]N/G[124]S/A39S 273 {GlaSwap FIX/ {Gla Swap FIX/ 274 E40L}/Q286R/M298Q E[40]L}/Q143R/M156Q {GlaSwap FIX/ {Gla Swap FIX/ 275 K43I}/Q286R/M298Q K[43]I}/Q143R/M156Q {GlaSwap FIX/ {Gla Swap FIX/ 276 Q44S}/Q286R/M298Q Q[44]S}/Q143R/M156Q {GlaSwap FIX/ {Gla Swap FIX/ 277 M19K}/Q286R/M298Q M[19]K}/Q143R/M156Q {GlaSwap FIX/ {GlaSwapFIX/M[19]K/E[40]L/K[43]I/ 278M19K/E40L/K43I/Q44S}/Q286R/M298Q Q[44]S}/Q143R/ M156Q T128N/P129A/Q286RT[128]N/P[129]A/Q143R 279 T128N/P129A/Q286R/M298QT[128]N/P[129]A/Q143R/M156Q 280 T128N/P129A/Q286R/H373FT[128]N/P[129]A/Q143R/H224F 281 V158D/Q286R/E296V/M298QV21D/Q143R/E154V/M156Q 282 T128N/P129A/V158D/E296V/M298QT[128]N/P[129]A/V21D/E154V/ 283 M156Q T128N/P129A/S222AT[128]N/P[129]A/S82A 284 GlaSwapFIX/T128N/P129A/S222A/GlaSwapFIX/T[128]N/P[129]A/S82A/ 285 Q286R Q143RGlaSwapFIX/T128N/P129A/Q286R/ GlaSwapFIX/T[128]N/P[129]A/Q143R/ 286M298Q M156Q T128N/P129A/S222A/H257A/Q286R/T[128]N/P[129]A/S82A/H117A/Q143R/ 287 M298Q M156QT128N/P129A/Q286R/M298Q/H373F T[128]N/P[129]A/Q143R/M156Q/ 288 H224FS52A/S60A/V158D/E296V/M298Q S[52]A/S[60]A/V21D/E154V/M156Q 289S52A/S60A/Q286R S[52]A/S[60]A/Q143R 290 S52A/S60A/S222AS[52]A/S[60]A/S82A 291 GlaSwapFIX/S52A/S60A/S222A/Q286RGlaSwapFIX/S[52]A/S[60]A/S82A/ 292 Q143R S52A/S60A/Q286R/M298QS[52]A/S[60]A/Q143R/M156Q 293 GlaSwapFIX/S52A/S60A/Q286R/M298QGlaSwapFIX/S[52]A/S[60]A/Q143R/ 294 M156Q S52A/S60A/S222A/H257A/Q286R/S[52]A/S[60]A/S82A/H117A/Q143R/ 298 M298Q M156Q S52A/S60A/Q286R/H373FS[52]A/S[60]A/Q143R/H224F 296 S52A/S60A/Q286R/M298Q/H373FS[52]A/S[60]A/Q143R/M156Q/H224F 297 V158D/T239V/E296V/M298QV21D/T99V/E154V/M156Q 298 T239V/Q286R T99V/Q143R 299 S222A/T239VS82A/T99V 300 Gla Swap FIX/S222A/T239V/Q286R Gla SwapFIX/S82A/T99V/Q143R 301 T239V/Q286R/M298Q T99V/Q143R/M156Q 302S222A/T239V/H257A/Q286R/M298Q S82A/T99V/H117A/Q143R/M156Q 303GlaSwapFIX/T239V/Q286R/M298Q GlaSwapFIX/T99V/Q143R/M156Q 304T239V/Q286R/H373F T99V/Q143R/H224F 305 T239V/Q286R/M298Q/H373FT99V/Q143R/M156Q/H224F 306 V158D/T239I/E296V/M298Q V21D/T99I/E154V/M156Q307 T239I/Q286R T99I/Q143R 308 S222A/T239I S82A/T99I 309GlaSwapFIX/S222A/T239I/Q286R GlaSwapFIX/S82A/T99I/Q143R 310T239I/Q286R/M298Q T99I/Q143R/M156Q 311 S222A/T239I/H257A/Q286R/M298QS82A/T99I/H117A/Q143R/M156Q 312 GlaSwapFIX/T239I/Q286R/M298QGlaSwapFIX/T99I/Q143R/M156Q 313 T239I/Q286R/H373F T99I/Q143R/H224F 314T239I/Q286R/M298Q/H373F T99I/Q143R/M156Q/H224F 315GlaSwapFIX/S222A/Q286R/H373F GlaSwapFIX/S82A/Q143R/H224F 316GlaSwapFIX/S222A/Q286R/M298Q GlaSwapFIX/S82A/Q143R/M156Q 317GlaSwapFIX/S222A/Q286R/M298Q/ GlaSwapFIX/S82A/Q143R/M156Q/ 318 H373FH224F V158D/E296V/M298Q/H373F V21D/E154V/M156Q/H224F 319V158D/Q286R/E296V/M298Q/H373F V21D/Q143R/E154V/M156Q/H224F 320H257A/Q286R/M298Q H117A/Q143R/M156Q 321 H257S/Q286R/M298QH117S/Q143R/M156Q 322 GlaSwapFIX/S222A/H257S/Q286RGlaSwapFIX/S82A/H117S/Q143R 323 S222A/H257S/Q286R/M298QS82A/H117S/Q143R/M156Q 324 H257S/Q286R/M298Q/H373FH117S/Q143R/M156Q/H224F 325 S222A/Q286R/M298Q/H373FS82A/Q143R/M156Q/H224F 326 GlaSwapFIX/Q366V GlaSwapFIX/Q217V 327S222A/Q286R/M298Q S82A/Q143R/M156Q 328 T128N/P129A/A175S/Q366VT[128]N/P[129]A/A39S/Q217V 329 A122N/G124S/A175S/Q366VA[122]N/G[124]S/A39S/Q217V 330 T128N/P129A/A175S/S222AT[128]N/P[129]A/A39S/S82A 331 A122N/G124S/A175S/S222AA[122]N/G[124]S/A39S/S82A 332 T128N/P129A/A175S/Q286RT[128]N/P[129]A/A39S/Q143R 333 A122N/G124S/A175S/Q286RA[122]N/G[124]S/A39S/Q143R 334 GlaSwapFIX/T128N/P129A/A175S/S222A/GlaSwapFIX/T[128]N/P[129]A/A39S/ 335 Q286R S82A/Q143RGlaSwapFIX/A122N/G124S/A175S/ GlaSwapFIX/A[122]N/G[124]S/A39S/ 336S222A/Q286R S82A/Q143R T128N/P129A/A175S/Q286R/M298QT[128]N/P[129]A/A39S/Q143R/M156Q 337 A122N/G124S/A175S/Q286R/M298QA[122]N/G[124]S/A39S/Q143R/ 338 M156Q T128N/P129A/A175S/S222A/H257A/T[128]N/P[129]A/A39S/S82A/H117A/ 339 Q286R/M298Q Q143R/M156QA122N/G124S/A175S/S222A/H257A/ A[122]N/G[124]S/A39S/S82A/H117A/ 340Q286R/M298Q Q143R/M156Q T128N/P129A/A175S/Q286R/M298Q/T[128]N/P[129]A/A39S/Q143R/M156Q/ 341 H373F H224FA122N/G124S/A175S/Q286R/M298Q/ A[122]N/G[124]S/A39S/Q143R/ 342 H373FM156Q/H224F T128N/P129A/M298Q T[128]N/P[129]A/M156Q 354 {Gla Swap FIX/{Gla Swap FIX/K[43]I}/ 355 K43I}/T128N/P129A/Q286R/M298QT[128]N/P[129]A/Q143R/M156Q T128N/P129A/Q286R/M298Q/Q366NT[128]N/P[129]A/Q143R/M156Q/ 356 Q217N {Gla Swap FIX/ {Gla Swap FIX/ 357K43I}/Q286R/M298Q/Q366N K[43]I}/Q143R/M156QQ217N {Gla Swap FIX/K43I}/{Gla Swap FIX/K[43]I}/ 358 T128N/P129A/Q286R/M298Q/Q366NT[128]N/P[129]A/Q143R/M156Q Q217N T128N/P129A/M298Q/H373FT[128]N/P[129]A/M156Q/H224F 359 V158D/Q286R/E296V/M298QV21D/Q143R/E154V/M156Q 360 M298Q/Q366N/H373F M156Q/Q217N/H224F 361T239V/M298Q/H373F T99V/M156Q/H224F 362 T239I/M298Q/H373FT99I/M156Q/H224F 363 T128N/P129A/Q286R/M298Q/Q366N/T[128]N/P[129]A/Q143R/M156Q/ 364 H373F Q217N/H224FT239V/Q286R/M298Q/Q366N T99V/Q143R/M156Q/Q217N 365T239I/Q286R/M298Q/Q366N T99I/Q143R/M156Q/Q217N 366T128N/P129A/T239V/Q286R/M298Q T[128]N/P[129]A/T99V/Q143R/M156Q 367T128N/P129A/S222A/T239V/H257A/ T[128]N/P[129]A/S82A/T99V/H117A/ 368Q286R/M298Q Q143R/M156Q T128N/P129A/T239V/Q286R/M298Q/T[128]N/P[129]A/T99V/Q143R/M156Q/ 369 H373F H224FT128N/P129A/T239I/Q286R/M298Q T[128]N/P[129]A/T99I/Q143R/M156Q 370T128N/P129A/T239I/Q286R/M298Q/ T[128]N/P[129]A/T99I/Q143R/M156Q/ 371H373F H224F

E. Production of FVII Polypeptides

FVII polypeptides, including modified FVII polypeptides, or domainsthereof of FVII or other vitamin-K polypeptide, can be obtained bymethods well known in the art for protein purification and recombinantprotein expression. Any method known to those of skill in the art foridentification of nucleic acids that encode desired genes can be used.Any method available in the art can be used to obtain a full length(i.e., encompassing the entire coding region) cDNA or genomic DNA cloneencoding a FVII polypeptide or other vitamin-K polypeptide, such as froma cell or tissue source, such as for example from liver. Modified FVIIpolypeptides can be engineered as described herein, such as bysite-directed mutagenesis.

FVII can be cloned or isolated using any available methods known in theart for cloning and isolating nucleic acid molecules. Such methodsinclude PCR amplification of nucleic acids and screening of libraries,including nucleic acid hybridization screening, antibody-based screeningand activity-based screening.

Methods for amplification of nucleic acids can be used to isolatenucleic acid molecules encoding a FVII polypeptide, including forexample, polymerase chain reaction (PCR) methods. A nucleic acidcontaining material can be used as a starting material from which aFVII-encoding nucleic acid molecule can be isolated. For example, DNAand mRNA preparations, cell extracts, tissue extracts (e.g. from liver),fluid samples (e.g. blood, serum, saliva), samples from healthy and/ordiseased subjects can be used in amplification methods. Nucleic acidlibraries also can be used as a source of starting material. Primers canbe designed to amplify a FVII-encoding molecule. For example, primerscan be designed based on expressed sequences from which a FVII isgenerated. Primers can be designed based on back-translation of a FVIIamino acid sequence. Nucleic acid molecules generated by amplificationcan be sequenced and confirmed to encode a FVII polypeptide.

Additional nucleotide sequences can be joined to a FVII-encoding nucleicacid molecule, including linker sequences containing restrictionendonuclease sites for the purpose of cloning the synthetic gene into avector, for example, a protein expression vector or a vector designedfor the amplification of the core protein coding DNA sequences.Furthermore, additional nucleotide sequences specifying functional DNAelements can be operatively linked to a FVII-encoding nucleic acidmolecule. Examples of such sequences include, but are not limited to,promoter sequences designed to facilitate intracellular proteinexpression, and secretion sequences designed to facilitate proteinsecretion. Additional nucleotide sequences such as sequences specifyingprotein binding regions also can be linked to FVII-encoding nucleic acidmolecules. Such regions include, but are not limited to, sequences tofacilitate uptake of FVII into specific target cells, or otherwiseenhance the pharmacokinetics of the synthetic gene.

The identified and isolated nucleic acids can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art can be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene, La Jolla, Calif.). The insertion into a cloning vector can,for example, be accomplished by ligating the DNA fragment into a cloningvector which has complementary cohesive termini. Insertion can beeffected using TOPO cloning vectors (Invitrogen, Carlsbad, Calif.). Ifthe complementary restriction sites used to fragment the DNA are notpresent in the cloning vector, the ends of the DNA molecules can beenzymatically modified. Alternatively, any site desired can be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers can contain specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and FVII proteingene can be modified by homopolymeric tailing. Recombinant molecules canbe introduced into host cells via, for example, transformation,transfection, infection, electroporation and sonoporation, so that manycopies of the gene sequence are generated.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated FVII protein gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene can be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

1. Vectors and Cells

For recombinant expression of one or more of the FVII proteins, thenucleic acid containing all or a portion of the nucleotide sequenceencoding the FVII protein can be inserted into an appropriate expressionvector, i.e., a vector that contains the necessary elements for thetranscription and translation of the inserted protein coding sequence.Exemplary of such a vector is any mammalian expression vector such as,for example, pCMV. The necessary transcriptional and translationalsignals also can be supplied by the native promoter for a FVII genes,and/or their flanking regions.

Also provided are vectors that contain nucleic acid encoding the FVII ormodified FVII. Cells containing the vectors also are provided. The cellsinclude eukaryotic and prokaryotic cells, and the vectors are anysuitable for use therein.

Prokaryotic and eukaryotic cells, including endothelial cells,containing the vectors are provided. Such cells include bacterial cells,yeast cells, fungal cells, Archea, plant cells, insect cells and animalcells. The cells are used to produce a FVII polypeptide or modified FVIIpolypeptide thereof by growing the above-described cells underconditions whereby the encoded FVII protein is expressed by the cell,and recovering the expressed FVII protein. For purposes herein, the FVIIcan be secreted into the medium.

In one embodiment, vectors containing a sequence of nucleotides thatencodes a polypeptide that has FVII activity and contains all or aportion of the FVII polypeptide, or multiple copies thereof, areprovided. The vectors can be selected for expression of the FVIIpolypeptide or modified FVII polypeptide thereof in the cell or suchthat the FVII protein is expressed as a secreted protein. When the FVIIis expressed the nucleic acid is linked to nucleic acid encoding asecretion signal, such as the Saccharomyces cerevisiae α-mating factorsignal sequence or a portion thereof, or the native signal sequence.

A variety of host-vector systems can be used to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g. vaccinia virus, adenovirus and otherviruses); insect cell systems infected with virus (e.g. baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements can be used.

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector can be used to construct expression vectorscontaining a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods can include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding a FVII polypeptide ormodified FVII polypeptide, or domains, derivatives, fragments orhomologs thereof, can be regulated by a second nucleic acid sequence sothat the genes or fragments thereof are expressed in a host transformedwith the recombinant DNA molecule(s). For example, expression of theproteins can be controlled by any promoter/enhancer known in the art. Ina specific embodiment, the promoter is not native to the genes for aFVII protein. Promoters which can be used include but are not limited tothe SV40 early promoter (Bernoist and Chambon, Nature 290:304-310(1981)), the promoter contained in the 3′ long terminal repeat of Roussarcoma virus (Yamamoto et al. Cell 22:787-797 (1980)), the herpesthymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA78:1441-1445 (1981)), the regulatory sequences of the metallothioneingene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expressionvectors such as the β-lactamase promoter (Jay et al., (1981) Proc. Natl.Acad. Sci. USA 78:5543) or the tac promoter (DeBoer et al., Proc. Natl.Acad. Sci. USA 80:21-25 (1983)); see also “Useful Proteins fromRecombinant Bacteria”: in Scientific American 242:79-94 (1980)); plantexpression vectors containing the nopaline synthetase promoter(Herrara-Estrella et al., Nature 303:209-213 (1984)) or the cauliflowermosaic virus 35S RNA promoter (Gardner et al., Nucleic Acids Res. 9:2871(1981)), and the promoter of the photosynthetic enzyme ribulosebisphosphate carboxylase (Herrera-Estrella et al., Nature 310:115-120(1984)); promoter elements from yeast and other fungi such as the Gal4promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinasepromoter, the alkaline phosphatase promoter, and the following animaltranscriptional control regions that exhibit tissue specificity and havebeen used in transgenic animals: elastase I gene control region which isactive in pancreatic acinar cells (Swift et al., Cell 38:639-646 (1984);Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986);MacDonald, Hepatology 7:425-515 (1987)); insulin gene control regionwhich is active in pancreatic beta cells (Hanahan et al., Nature315:115-122 (1985)), immunoglobulin gene control region which is activein lymphoid cells (Grosschedl et al., Cell 38:647-658 (1984); Adams etal., Nature 318:533-538 (1985); Alexander et al., Mol. Cell. Biol.7:1436-1444 (1987)), mouse mammary tumor virus control region which isactive in testicular, breast, lymphoid and mast cells (Leder et al.,Cell 45:485-495 (1986)), albumin gene control region which is active inliver (Pinckert et al., Genes and Devel. 1:268-276 (1987)),alpha-fetoprotein gene control region which is active in liver (Krumlaufet al., Mol. Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science235:53-58 1987)), alpha-1 antitrypsin gene control region which isactive in liver (Kelsey et al., Genes and Devel. 1:161-171 (1987)), betaglobin gene control region which is active in myeloid cells (Magram etal., Nature 315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)),myelin basic protein gene control region which is active inoligodendrocyte cells of the brain (Readhead et al., Cell 48:703-712(1987)), myosin light chain-2 gene control region which is active inskeletal muscle (Shani, Nature 314:283-286 (1985)), and gonadotrophicreleasing hormone gene control region which is active in gonadotrophs ofthe hypothalamus (Mason et al., Science 234:1372-1378 (1986)).

In a specific embodiment, a vector is used that contains a promoteroperably linked to nucleic acids encoding a FVII polypeptide or modifiedFVII polypeptide, or a domain, fragment, derivative or homolog, thereof,one or more origins of replication, and optionally, one or moreselectable markers (e.g., an antibiotic resistance gene). Vectors andsystems for expression of FVII polypeptides include the well knownPichia vectors (available, for example, from Invitrogen, San Diego,Calif.), particularly those designed for secretion of the encodedproteins. Exemplary plasmid vectors for expression in mammalian cellsinclude, for example, pCMV. Exemplary plasmid vectors for transformationof E. coli cells, include, for example, the pQE expression vectors(available from Qiagen, Valencia, Calif.; see also literature publishedby Qiagen describing the system). pQE vectors have a phage T5 promoter(recognized by E. coli RNA polymerase) and a double lac operatorrepression module to provide tightly regulated, high-level expression ofrecombinant proteins in E. coli, a synthetic ribosomal binding site (RBSII) for efficient translation, a 6×His tag coding sequence, t₀ and T1transcriptional terminators, ColE1 origin of replication, and abeta-lactamase gene for conferring ampicillin resistance. The pQEvectors enable placement of a 6×His tag at either the N- or C-terminusof the recombinant protein. Such plasmids include pQE 32, pQE 30, andpQE 31 which provide multiple cloning sites for all three reading framesand provide for the expression of N-terminally 6×His-tagged proteins.Other exemplary plasmid vectors for transformation of E. coli cells,include, for example, the pET expression vectors (see, U.S. Pat. No.4,952,496; available from NOVAGEN, Madison, Wis.; see, also literaturepublished by Novagen describing the system). Such plasmids include pET11a, which contains the T7lac promoter, T7 terminator, the inducible E.coli lac operator, and the lac repressor gene; pET 12a-c, which containsthe T7 promoter, T7 terminator, and the E. coli ompT secretion signal;and pET 15b and pET19b (NOVAGEN, Madison, Wis.), which contain aHis-Tag™ leader sequence for use in purification with a His column and athrombin cleavage site that permits cleavage following purification overthe column, the T7-lac promoter region and the T7 terminator.

2. Expression Systems

FVII polypeptides (modified and unmodified) can be produced by anymethods known in the art for protein production including in vitro andin vivo methods such as, for example, the introduction of nucleic acidmolecules encoding FVII into a host cell, host animal and expressionfrom nucleic acid molecules encoding FVII in vitro. FVII and modifiedFVII polypeptides can be expressed in any organism suitable to producethe required amounts and forms of a FVII polypeptide needed foradministration and treatment. Expression hosts include prokaryotic andeukaryotic organisms such as E. coli, yeast, plants, insect cells,mammalian cells, including human cell lines and transgenic animals.Expression hosts can differ in their protein production levels as wellas the types of post-translational modifications that are present on theexpressed proteins. The choice of expression host can be made based onthese and other factors, such as regulatory and safety considerations,production costs and the need and methods for purification.

Expression in eukaryotic hosts can include expression in yeasts such asSaccharomyces cerevisiae and Pichia pastoris, insect cells such asDrosophila cells and lepidopteran cells, plants and plant cells such astobacco, corn, rice, algae, and lemna. Eukaryotic cells for expressionalso include mammalian cells lines such as Chinese hamster ovary (CHO)cells or baby hamster kidney (BHK) cells. Eukaryotic expression hostsalso include production in transgenic animals, for example, includingproduction in serum, milk and eggs. Transgenic animals for theproduction of wild-type FVII polypeptides are known in the art (U.S.Patent Publication Nos. 20020166130 and 20040133930) and can be adaptedfor production of modified FVII polypeptides provided herein.

Many expression vectors are available and known to those of skill in theart for the expression of FVII. The choice of expression vector isinfluenced by the choice of host expression system. Such selection iswell within the level of skill of the skilled artisan. In general,expression vectors can include transcriptional promoters and optionallyenhancers, translational signals, and transcriptional and translationaltermination signals. Expression vectors that are used for stabletransformation typically have a selectable marker which allows selectionand maintenance of the transformed cells. In some cases, an origin ofreplication can be used to amplify the copy number of the vectors in thecells.

FVII or modified FVII polypeptides also can be utilized or expressed asprotein fusions. For example, a fusion can be generated to addadditional functionality to a polypeptide. Examples of fusion proteinsinclude, but are not limited to, fusions of a signal sequence, a tagsuch as for localization, e.g. a his₆ tag or a myc tag, or a tag forpurification, for example, a GST fusion, and a sequence for directingprotein secretion and/or membrane association.

In one embodiment, the FVII polypeptide or modified FVII polypeptidescan be expressed in an active form, whereby activation is achieved byautoactivation of the polypeptide following secretion. In anotherembodiment, the protease is expressed in an inactive, zymogen form.

Methods of production of FVII polypeptides can include coexpression ofone or more additional heterologous polypeptides that can aid in thegeneration of the FVII polypeptides. For example, such polypeptides cancontribute to the post-translation processing of the FVII polypeptides.Exemplary polypeptides include, but are not limited to, peptidases thathelp cleave FVII precursor sequences, such as the propeptide sequence,and enzymes that participate in the modification of the FVIIpolypeptide, such as by glycosylation, hydroxylation, carboxylation, orphosphorylation, for example. An exemplary peptidase that can becoexpressed with FVII is PACE/furin (or PACE-SOL), which aids in thecleavage of the FVII propeptide sequence. An exemplary protein that aidsin the carboxylation of the FVII polypeptide is the warfarin-sensitiveenzyme vitamin K 2,3-epoxide reductase (VKOR), which produces reducedvitamin K for utilization as a cofactor by the vitamin K-dependentγ-carboxylase (Wajih et al., J. Biol. Chem. 280(36)31603-31607). Asubunit of this enzyme, VKORC1, can be coexpressed with the modifiedFVII polypeptide to increase the γ-carboxylation The one or moreadditional polypeptides can be expressed from the same expression vectoras the FVII polypeptide or from a different vector.

a. Prokaryotic Expression

Prokaryotes, especially E. coli, provide a system for producing largeamounts of FVII (see, for example, Platis et al. (2003) Protein Exp.Purif. 31(2): 222-30; and Khalilzadeh et al. (2004) J. Ind. Microbiol.Biotechnol. 31(2): 63-69). Transformation of E. coli is a simple andrapid technique well known to those of skill in the art. Expressionvectors for E. coli can contain inducible promoters that are useful forinducing high levels of protein expression and for expressing proteinsthat exhibit some toxicity to the host cells. Examples of induciblepromoters include the lac promoter, the trp promoter, the hybrid tacpromoter, the T7 and SP6 RNA promoters and the temperature regulatedλP_(L) promoter.

FVII can be expressed in the cytoplasmic environment of E. coli. Thecytoplasm is a reducing environment and for some molecules, this canresult in the formation of insoluble inclusion bodies. Reducing agentssuch as dithiothreitol and β-mercaptoethanol and denaturants (e.g., suchas guanidine-HCl and urea) can be used to resolubilize the proteins. Analternative approach is the expression of FVII in the periplasmic spaceof bacteria which provides an oxidizing environment and chaperonin-likeand disulfide isomerases leading to the production of soluble protein.Typically, a leader sequence is fused to the protein to be expressedwhich directs the protein to the periplasm. The leader is then removedby signal peptidases inside the periplasm. Examples ofperiplasmic-targeting leader sequences include the pelB leader from thepectate lyase gene and the leader derived from the alkaline phosphatasegene. In some cases, periplasmic expression allows leakage of theexpressed protein into the culture medium. The secretion of proteinsallows quick and simple purification from the culture supernatant.Proteins that are not secreted can be obtained from the periplasm byosmotic lysis. Similar to cytoplasmic expression, in some cases proteinscan become insoluble and denaturants and reducing agents can be used tofacilitate solubilization and refolding. Temperature of induction andgrowth also can influence expression levels and solubility. Typically,temperatures between 25° C. and 37° C. are used. Mutations also can beused to increase solubility of expressed proteins. Typically, bacteriaproduce aglycosylated proteins. Thus, if proteins require glycosylationfor function, glycosylation can be added in vitro after purificationfrom host cells.

b. Yeast

Yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe,Yarrowia lipolytica, Kluyveromyces lactis, and Pichia pastoris areuseful expression hosts for FVII (see for example, Skoko et al. (2003)Biotechnol. Appl. Biochem. 38(Pt3):257-65). Yeast can be transformedwith episomal replicating vectors or by stable chromosomal integrationby homologous recombination. Typically, inducible promoters are used toregulate gene expression. Examples of such promoters include GAL1, GAL7,and GAL5 and metallothionein promoters such as CUP1. Expression vectorsoften include a selectable marker such as LEU2, TRP1, HIS3, and URA3 forselection and maintenance of the transformed DNA. Proteins expressed inyeast are often soluble and co-expression with chaperonins, such as Bipand protein disulfide isomerase, can improve expression levels andsolubility. Additionally, proteins expressed in yeast can be directedfor secretion using secretion signal peptide fusions such as the yeastmating type alpha-factor secretion signal from Saccharomyces cerevisiaeand fusions with yeast cell surface proteins such as the Aga2p matingadhesion receptor or the Arxula adeninivorans glucoamylase. A proteasecleavage site (e.g., the Kex-2 protease) can be engineered to remove thefused sequences from the polypeptides as they exit the secretionpathway. Yeast also is capable of glycosylation at Asn-X-Ser/Thr motifs.

c. Insects and Insect Cells

Insects and insect cells, particularly using a baculovirus expressionsystem, are useful for expressing polypeptides such as FVII or modifiedforms thereof (see, for example, Muneta et al. (2003) J. Vet. Med. Sci.65(2):219-23). Insect cells and insect larvae, including expression inthe haemolymph, express high levels of protein and are capable of mostof the post-translational modifications used by higher eukaryotes.Baculoviruses have a restrictive host range which improves the safetyand reduces regulatory concerns of eukaryotic expression. Typically,expression vectors use a promoter such as the polyhedrin promoter ofbaculovirus for high level expression. Commonly used baculovirus systemsinclude baculoviruses such as Autographa californica nuclearpolyhedrosis virus (AcNPV), and the Bombyx mori nuclear polyhedrosisvirus (BmNPV) and an insect cell line such as Sf9 derived fromSpodoptera frugiperda, Pseudaletia unipuncta (A7S) and Danaus plexippus(DpN1). For high level expression, the nucleotide sequence of themolecule to be expressed is fused immediately downstream of thepolyhedrin initiation codon of the virus. Mammalian secretion signalsare accurately processed in insect cells and can be used to secrete theexpressed protein into the culture medium. In addition, the cell linesPseudaletia unipuncta (A7S) and Danaus plexippus (DpN1) produce proteinswith glycosylation patterns similar to mammalian cell systems.

An alternative expression system in insect cells is the use of stablytransformed cells. Cell lines such as the Schnieder 2 (S2) and Kc cells(Drosophila melanogaster) and C7 cells (Aedes albopictus) can be usedfor expression. The Drosophila metallothionein promoter can be used toinduce high levels of expression in the presence of heavy metalinduction with cadmium or copper. Expression vectors are typicallymaintained by the use of selectable markers such as neomycin andhygromycin.

d. Mammalian Cells

Mammalian expression systems can be used to express FVII polypeptides.Expression constructs can be transferred to mammalian cells by viralinfection such as adenovirus or by direct DNA transfer such asliposomes, calcium phosphate, DEAE-dextran and by physical means such aselectroporation and microinjection. Expression vectors for mammaliancells typically include an mRNA cap site, a TATA box, a translationalinitiation sequence (Kozak consensus sequence) and polyadenylationelements. Such vectors often include transcriptional promoter-enhancersfor high level expression, for example the SV40 promoter-enhancer, thehuman cytomegalovirus (CMV) promoter, and the long terminal repeat ofRous sarcoma virus (RSV). These promoter-enhancers are active in manycell types. Tissue and cell-type promoters and enhancer regions also canbe used for expression. Exemplary promoter/enhancer regions include, butare not limited to, those from genes such as elastase I, insulin,immunoglobulin, mouse mammary tumor virus, albumin, alpha-fetoprotein,alpha 1-antitrypsin, beta-globin, myelin basic protein, myosin lightchain-2, and gonadotropic releasing hormone gene control. Selectablemarkers can be used to select for and maintain cells with the expressionconstruct. Examples of selectable marker genes include, but are notlimited to, hygromycin B phosphotransferase, adenosine deaminase,xanthine-guanine phosphoribosyl transferase, aminoglycosidephosphotransferase, dihydrofolate reductase and thymidine kinase. Fusionwith cell surface signaling molecules such as TCR-ξ and Fc_(ε)RI-γ candirect expression of the proteins in an active state on the cellsurface.

Many cell lines are available for mammalian expression including mouse,rat human, monkey, and chicken and hamster cells. Exemplary cell linesinclude, but are not limited to, BHK (i.e. BHK-21 cells), 293-F, CHO,Balb/3T3, HeLa, MT2, mouse NSO (non-secreting) and other myeloma celllines, hybridoma and heterohybridoma cell lines, lymphocytes,fibroblasts, Sp2/0, COS, NIH3T3, HEK293, 293S, 293T, 2B8, and HKB cells.Cell lines also are available adapted to serum-free media whichfacilitates purification of secreted proteins from the cell culturemedia. One such example is the serum free EBNA-1 cell line (Pham et al.,(2003) Biotechnol. Bioeng. 84:332-42). Expression of recombinant FVIIpolypeptides exhibiting similar structure and post-translationalmodifications as plasma-derived FVII are known in the art (see, e.g.,Jurlander et al. (2001) Semin Throm Hemost 27:373-384). Methods ofoptimizing vitamin K-dependent protein expression are known. Forexample, supplementation of vitamin K in culture medium or co-expressionof vitamin K-dependent γ-carboxylases (Wajih et al., J. Biol. Chem.280(36)31603-31607) can aid in post-translational modification ofvitamin K-dependent proteins, such as FVII polypeptides.

e. Plants

Transgenic plant cells and plants can be used for the expression ofFVII. Expression constructs are typically transferred to plants usingdirect DNA transfer such as microprojectile bombardment and PEG-mediatedtransfer into protoplasts, and with agrobacterium-mediatedtransformation. Expression vectors can include promoter and enhancersequences, transcriptional termination elements, and translationalcontrol elements. Expression vectors and transformation techniques areusually divided between dicot hosts, such as Arabidopsis and tobacco,and monocot hosts, such as corn and rice. Examples of plant promotersused for expression include the cauliflower mosaic virus promoter, thenopaline synthase promoter, the ribose bisphosphate carboxylase promoterand the ubiquitin and UBQ3 promoters. Selectable markers such ashygromycin, phosphomannose isomerase and neomycin phosphotransferase areoften used to facilitate selection and maintenance of transformed cells.Transformed plant cells can be maintained in culture as cells,aggregates (callus tissue) or regenerated into whole plants. Becauseplants have different glycosylation patterns than mammalian cells, thiscan influence the choice to produce FVII in these hosts. Transgenicplant cells also can include algae engineered to produce proteins (see,for example, Mayfield et al. (2003) PNAS 100:438-442). Because plantshave different glycosylation patterns than mammalian cells, this caninfluence the choice to produce FVII in these hosts.

2. Purification

Methods for purification of FVII polypeptides from host cells depend onthe chosen host cells and expression systems. For secreted molecules,proteins are generally purified from the culture media after removingthe cells. For intracellular expression, cells can be lysed and theproteins purified from the extract. When transgenic organisms such astransgenic plants and animals are used for expression, tissues or organscan be used as starting material to make a lysed cell extract.Additionally, transgenic animal production can include the production ofpolypeptides in milk or eggs, which can be collected, and if necessaryfurther the proteins can be extracted and further purified usingstandard methods in the art.

FVII can be purified using standard protein purification techniquesknown in the art including but not limited to, SDS-PAGE, size fractionand size exclusion chromatography, ammonium sulfate precipitation,chelate chromatography and ionic exchange chromatography. For example,FVII polypeptides can be purified by anion exchange chromatography.Exemplary of a method to purify FVII polypeptides is by using an ionexchange column that permits binding of any polypeptide that has afunctional Gla domain, followed by elution in the presence of calcium(See e.g., Example 2). Affinity purification techniques also can be usedto improve the efficiency and purity of the preparations. For example,antibodies, receptors and other molecules that bind FVII can be used inaffinity purification. In another example, purification also can beenhanced using a soluble TF (sTF) affinity column (Maun et al. (2005)Prot Sci 14:1171-1180). Expression constructs also can be engineered toadd an affinity tag such as a myc epitope, GST fusion or His₆ andaffinity purified with myc antibody, glutathione resin, and Ni-resin,respectively, to a protein. Purity can be assessed by any method knownin the art including gel electrophoresis and staining andspectrophotometric techniques.

The FVII protease can be expressed and purified to be in an inactiveform (zymogen form) or alternatively the expressed protease can bepurified into an active form, such as by autocatalysis. For example,FVII polypeptides that have been activated via proteolytic cleavage ofthe Arg¹⁵²-Ile¹⁵³ can be prepared in vitro (i.e. FVIIa; two-chain form).The FVII polypeptides can be first prepared by any of the methods ofproduction described herein, including, but not limited to, productionin mammalian cells followed by purification. Cleavage of the FVIIpolypeptides into the active protease form, FVIIa, can be accomplishedby several means. For example, autoactivation during incubation withphospholipid vesicles in the presence of calcium can be achieved in 45minutes (Nelsestuen et al. (2001) J Biol Chem 276:39825-31). FVIIpolypeptides also can be activated to completion by incubation withfactor Xa, factor XIIa or TF in the presence calcium, with or withoutphospholipids (see e.g., Example 2 and Broze et al. (1980) J Biol Chem255:1242-1247, Higashi et al. (1996) J Biol Chem 271:26569-26574, Harveyet al. J Biol Chem 278:8363-8369).

3. Fusion Proteins

Fusion proteins containing a modified FVII polypeptide and one or moreother polypeptides also are provided. Pharmaceutical compositionscontaining such fusion proteins formulated for administration by asuitable route are provided. Fusion proteins are formed by linking inany order the modified FVII polypeptide and an agent, such as anantibody or fragment thereof, growth factor, receptor, ligand, and othersuch agent for the purposes of facilitating the purification of a FVIIpolypeptide, altering the pharmacodynamic properties of a FVIIpolypeptide by directing, for example, by directing the polypeptide to atargeted cell or tissue, and/or increasing the expression or secretionof the FVII polypeptide. Typically any FVII fusion protein retains atleast about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% coagulant activitycompared with a non-fusion FVII polypeptide, including 96%, 97%, 98%,99% or greater coagulant activity compared with a non-fusionpolypeptide.

Linkage of a FVII polypeptide with another polypeptide can be effecteddirectly or indirectly via a linker. In one example, linkage can be bychemical linkage, such as via heterobifunctional agents or thiollinkages or other such linkages. Fusion also can be effected byrecombinant means. Fusion of a FVII polypeptide to another polypeptidecan be to the N- or C-terminus of the FVII polypeptide. Non-limitingexamples of polypeptides that can be used in fusion proteins with a FVIIpolypeptide provided herein include, for example, a GST (glutathioneS-transferase) polypeptide, Fc domain from immunoglobulin G, or aheterologous signal sequence. The fusion proteins can contain additionalcomponents, such as E. coli maltose binding protein (MBP) that aid inuptake of the protein by cells (see, International PCT application No.WO 01/32711).

A fusion protein can be produced by standard recombinant techniques. Forexample, DNA fragments coding for the different polypeptide sequencescan be ligated together in-frame in accordance with conventionaltechniques, e.g., by employing blunt-ended or stagger-ended termini forligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers that give rise to complementary overhangs betweentwo consecutive gene fragments that can subsequently be annealed andreamplified to generate a chimeric gene sequence (see, e.g., Ausubel etal. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons,1992). Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). AFVII-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the protease protein.

4. Polypeptide Modification

Modified FVII polypeptides can be prepared as naked polypeptide chainsor as a complex. For some applications, it can be desirable to preparemodified FVII in a “naked” form without post-translational or otherchemical modifications. Naked polypeptide chains can be prepared insuitable hosts that do not post-translationally modify FVII. Suchpolypeptides also can be prepared in in vitro systems and using chemicalpolypeptide synthesis. For other applications, particular modificationscan be desired including pegylation, albumination, glycosylation,carboxylation, hydroxylation, phosphorylation, or other knownmodifications. Modifications can be made in vitro or, for example, byproducing the modified FVII in a suitable host that produces suchmodifications.

5. Nucleotide Sequences

Nucleic acid molecules encoding FVII or modified FVII polypeptides areprovided herein. Nucleic acid molecules include allelic variants orsplice variants of any encoded FVII polypeptide. Exemplary of nucleicacid molecules provided herein are any that encode a modified FVIIpolypeptide provided herein, such as any encoding a polypeptide setforth in any of SEQ ID NOS: 113-273. In one embodiment, nucleic acidmolecules provided herein have at least 50, 60, 65, 70, 75, 80, 85, 90,91, 92, 93, 94, 95, or 99% sequence identity or hybridize underconditions of medium or high stringency along at least 70% of thefull-length of any nucleic acid encoding a FVII polypeptide providedherein. In another embodiment, a nucleic acid molecule can include thosewith degenerate codon sequences encoding any of the FVII polypeptidesprovided herein.

F. Assessing Modified FVII Polypeptide Activities

The activities and properties of FVII polypeptides can be assessed invitro and/or in vivo. Assays for such assessment are known to those ofskill in the art and are known to correlate tested activities andresults to therapeutic and in vivo activities. In one example, FVIIvariants can be assessed in comparison to unmodified and/or wild-typeFVII. In another example, the activity of modified FVII polypeptides canbe assessed following exposure in vitro or in vivo to AT-III andcompared with that of modified FVII polypeptides that have not beenexposed to AT-III. Such assays can be performed in the presence orabsence of TF. In vitro assays include any laboratory assay known to oneof skill in the art, such as for example, cell-based assays includingcoagulation assays, binding assays, protein assays, and molecularbiology assays. In vivo assays include FVII assays in animal models aswell as administration to humans. In some cases, activity of FVII invivo can be determined by assessing blood, serum, or other bodily fluidfor assay determinants. FVII variants also can be tested in vivo toassess an activity or property, such as therapeutic effect.

Typically, assays described herein are with respect to the two-chainactivated form of FVII, i.e. FVIIa. Such assays also can be performedwith the single chain form, such as to provide a negative control sincesuch form typically does not contain proteolytic or catalytic activityrequired for the coagulant activity of FVII. In addition, such assaysalso can be performed in the presence of cofactors, such as TF, which insome instances augments the activity of FVII.

1. In Vitro Assays

Exemplary in vitro assays include assays to assess polypeptidemodification and activity. Modifications can be assessed using in vitroassays that assess γ-carboxylation and other post-translationalmodifications, protein assays and conformational assays known in theart. Assays for activity include, but are not limited to, measurement ofFVII interaction with other coagulation factors, such as TF, factor Xand factor IX, proteolytic assays to determine the proteolytic activityof FVII polypeptides, assays to determine the binding and/or affinity ofFVII polypeptides for phosphatidylserines and other phospholipids, andcell based assays to determine the effect of FVII polypeptides oncoagulation.

Concentrations of modified FVII polypeptides can be assessed by methodswell-known in the art, including but not limited to, enzyme-linkedimmunosorbant assays (ELISA), SDS-PAGE; Bradford, Lowry, BCA methods; UVabsorbance, and other quantifiable protein labeling methods, such as,but not limited to, immunological, radioactive and fluorescent methodsand related methods.

Assessment of cleavage products of proteolysis reactions, includingcleavage of FVII polypeptides or products produced by FVII proteaseactivity, can be performed using methods including, but not limited to,chromogenic substrate cleavage, HPLC, SDS-PAGE analysis, ELISA, Westernblotting, immunohistochemistry, immunoprecipitation, NH2-terminalsequencing, and protein labeling.

Structural properties of modified FVII polypeptides can also beassessed. For example, X-ray crystallography, nuclear magnetic resonance(NMR), and cryoelectron microscopy (cryo-EM) of modified FVIIpolypeptides can be performed to assess three-dimensional structure ofthe FVII polypeptides and/or other properties of FVII polypeptides, suchas Ca²⁺ or cofactor binding.

Additionally, the presence and extent of FVII degradation can bemeasured by standard techniques such as sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting ofelectrophoresed FVII-containing samples. FVII polypeptides that havebeen exposed to proteases can also be subjected to N-terminal sequencingto determine location or changes in cleavage sites of the modified FVIIpolypeptides.

a. Post-Translational Modification

FVII polypeptides also can be assessed for the presence ofpost-translational modifications. Such assays are known in the art andinclude assays to measure glycosylation, hydroxylation, andcarboxylation. In an exemplary assay for glycosylation, carbohydrateanalysis can be performed, for example, with SDS page analysis of FVIIpolypeptides exposed to hydrazinolysis or endoglycosidase treatment.Hydrazinolysis releases N- and O-linked glycans from glycoproteins byincubation with anhydrous hydrazine, while endoglycosidase releaseinvolves PNGase F, which releases most N-glycans from glycoproteins.Hydrazinolysis or endoglycosidase treatment of FVII polypeptidesgenerates a reducing terminus that can be tagged with a fluorophore orchromophore label. Labeled FVII polypeptides can be analyzed byfluorophore-assisted carbohydrate electrophoresis (FACE). Thefluorescent tag for glycans also can be used for monosaccharideanalysis, profiling or fingerprinting of complex glycosylation patternsby HPLC. Exemplary HPLC methods include hydrophilic interactionchromatography, electronic interaction, ion-exchange, hydrophobicinteraction, and size-exclusion chromatography. Exemplary glycan probesinclude, but are not limited to, 3-(acetylamino)-6-aminoacridine (AA-Ac)and 2-aminobenzoic acid (2-AA). Carbohydrate moieties can also bedetected through use of specific antibodies that recognize theglycosylated FVII polypeptide. An exemplary assay to measureβ-hydroxylation comprises reverse phase HPLC analysis of FVIIpolypeptides that have been subjected to alkaline hydrolysis (Przysieckiet al. (1987) PNAS 84:7856-7860). Carboxylation and γ-carboxylation ofFVII polypeptides can be assessed using mass spectrometry withmatrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)analysis, as described in the art (se, e.g. Harvey et al. J Biol Chem278:8363-8369, Maun et al. Prot Sci 14:1171-1180). The interaction of aFVII polypeptide containing the propeptide (pro-FVII) with thecarboxylase responsible for post-translational γ-carboxylatemodification also can be assessed. The dissociation constant (K_(d))following incubation of carboxylase with flourescin-labeled pro-FVIIpolypeptides can be measured by determining the amount of boundcarboxylase by anisotropy (Lin et al. (2004) J Biol Chem 279:6560-6566).

b. Proteolytic Activity

Modified FVII polypeptides can be tested for proteolytic activity. Theproteolytic activity of FVII can be measured using chromogenicsubstrates such as Chromozym t-PA (MeSO₂-D-Phe-Gly-Arg-pNA), S-2288(H-D-Ile-Pro-Arg-pNA), S-2266 (H-D-Val-Leu-Arg-pNA), S-2765(Z-D-Arg-Gly-Arg-pNA), Spectrozyme FXa and Spectrozyme FVIIa(CH₃SO₂-D-CHA-But-Arg-pNA). FVII polypeptides, alone or in the presenceof TF, are incubated with varying concentrations of chromogenicsubstrate. Cleavage of the substrate can be monitored by absorbance andthe rate of substrate hydrolysis determined by linear regression usingsoftware readily available.

The activation of coagulation factor substrates, such as FX, by FVIIpolypeptides also can be assessed. FVII polypeptides, with or withoutpreincubation with TF, can be incubated with purified FX (availablecommercially). The amount of active FXa produced as a consequence ofincubation with FVII polypeptides is measured as activity of FXa for achromogenic substrate, such as S-2222 or Spectrafluor FXa(CH₃SO₂-D-CHA-Gly-Arg-AMC.AcOH), which is monitored via absorbancechanges (Harvey et al. J Biol Chem 278:8363-8369, see also Example 4below). A source of phospholipid also can be included in the incubationof FVII and FX (Nelsestuen et al. (2001) Biol Chem 276:39825-31).

c. Coagulation Activity

FVII polypeptides can be tested for coagulation activity by using assayswell known in the art. For example, some of the assays include, but arenot limited to, a two stage clotting assay (Liebman et al., (1985) PNAS82:3879-3883); the prothrombin time assay (PT, which can measureTF-dependent activity of FVIIa in the extrinsic pathway); assays whichare modifications of the PT test; the activated partial thromboplastintime (aPTT, which can measure TF-independent activity of FVIIa);activated clotting time (ACT); recalcified activated clotting time; theLee-White Clotting time; or thromboelastography (TEG) (Pusateri et al.(2005) Critical Care 9:S15-S24). For example, coagulation activity of amodified FVII polypeptide can be determined by a PT-based assay whereFVII is diluted in FVII-deficient plasma, and mixed with prothrombintime reagent (recombinant TF with phospholipids and calcium), such asthat available as Innovin™ from Dade Behring. Clot formation is detectedoptically and time to clot is determined and compared againstFVII-deficient plasma alone.

d. Binding to and/or Inhibition by Other Proteins and Molecules

Inhibition assays can be used to measure resistance of modified FVIIpolypeptides to FVII inhibitors, such as, for example, AT-III and TFPI,or molecules such as Zn²⁺. Assessment of inhibition to other inhibitorsalso can be tested and include, but are not limited to, other serineprotease inhibitors, and FVII-specific antibodies. Inhibition can beassessed by incubation of, for example, AT-III, TFPI or Zn²⁺ with FVIIpolypeptides that have been preincubated with and/or without TF. Theactivity of FVII can then be measured using any one or more of theactivity or coagulation assays described above, and inhibition byAT-III, TFPI, or Zn²⁺ can be assessed by comparing the activity of FVIIpolypeptides incubated with the inhibitor, with the activity of FVIIpolypeptides that were not incubated with the inhibitor.

FVII polypeptides can be tested for binding to other coagulation factorsand inhibitors. For example, FVII direct and indirect interactions withcofactors, such as TF, substrates, such as FX and FIX, and inhibitors,such as antithrombin III, TFPI, and heparin can be assessed using anybinding assay known in the art, including, but not limited to,immunoprecipitation, column purification, non-reducing SDS-PAGE,BIAcore® assays, surface plasmon resonance (SPR), fluorescence resonanceenergy transfer (FRET), fluorescence polarization (FP), isothermaltitration calorimetry (ITC), circular dichroism (CD), protein fragmentcomplementation assays (PCA), Nuclear Magnetic Resonance (NMR)spectroscopy, light scattering, sedimentation equilibrium, small-zonegel filtration chromatography, gel retardation, Far-western blotting,fluorescence polarization, hydroxyl-radical protein footprinting, phagedisplay, and various two-hybrid systems. In one example, Zn²⁺ binding isassessed using equilibrium analysis (Petersen et al., (2000) ProteinScience 9:859-866)

e. Phospholipid Affinity

Modified FVII polypeptide binding and/or affinity for phosphatidylserine(PS) and other phospholipids can be determined using assays well knownin the art. Highly pure phospholipids (for example, known concentrationsof bovine PS and egg phosphatidylcholine (PC), which are commerciallyavailable, such as from Sigma, in organic solvent can be used to preparesmall unilamellar phospholipid vesicles. FVII polypeptide binding tothese PS/PC vesicles can be determined by relative light scattering at900 to the incident light. The intensity of the light scatter with PC/PSalone and with PC/PS/FVII is measured to determine the dissociationconstant (Harvey et al. J Biol Chem 278:8363-8369). Surface plasmaresonance, such as on a BIAcore biosensor instrument, also can be usedto measure the affinity of FVII polypeptides for phospholipid membranes(Sun et al. Blood 101:2277-2284).

2. Non-Human Animal Models

Non-human animal models can be used to assess activity, efficacy andsafety of modified FVII polypeptides. For example, non-human animals canbe used as models for a disease or condition. Non-human animals can beinjected with disease and/or phenotype-inducing substances prior toadministration of FVII variants, such as any FVII variant set forth inany of SEQ ID NOS: 113-273, to monitor the effects on diseaseprogression. Genetic models also are useful. Animals, such as mice, canbe generated which mimic a disease or condition by the overexpression,underexpression or knock-out of one or more genes, such as, for example,factor VIII knock-out mice that display hemophilia A (Bi et al. (1995)Nat Gen 10:119-121). Such animals can be generated by transgenic animalproduction techniques well-known in the art or using naturally-occurringor induced mutant strains. Examples of useful non-human animal models ofdiseases associated with FVII include, but are not limited to, models ofbleeding disorders, in particular hemophilia, or thrombotic disease.Non-human animal models for injury also can be used to assess anactivity, such as the coagulation activity, of FVII polypeptides. Thesenon-human animal models can be used to monitor activity of FVII variantscompared to a wild type FVII polypeptide.

Animal models also can be used to monitor stability, half-life, andclearance of modified FVII polypeptides. Such assays are useful forcomparing modified FVII polypeptides and for calculating doses and doseregimens for further non-human animal and human trials. For example, amodified FVII polypeptide, such as any FVII variant provided hereinincluding, for example, any set forth in any of SEQ ID NOS:113-273, canbe injected into the tail vein of mice. Blood samples are then taken attime-points after injection (such as minutes, hours and days afterwards)and then the level of the modified FVII polypeptides in bodily samplesincluding, but not limited to, serum or plasma can be monitored atspecific time-points for example by ELISA or radioimmunoassay. Bloodsamples from various time points following injection of the FVIIpolypeptides also be tested for coagulation activity using variousmethods, such as is described in Example 9. These types ofpharmacokinetic studies can provide information regarding half-life,clearance and stability of the FVII polypeptides, which can assist indetermining suitable dosages for administration as a procoagulant.

Modified FVII polypeptides, such as any set forth in any of SEQ ID NOS:113-273, can be tested for therapeutic effectiveness using animal modelsfor hemophilia. In one non-limiting example, an animal model such as amouse can be used. Mouse models of hemophilia are available in the artand can be employed to test modified FVII polypeptides. For example, amouse model of hemophilia A that is produced by injection withanti-FVIII antibodies can be used to assess the coagulant activity ofFVII polypeptides (see e.g. Example 6, and Tranholm et al. Blood(2003)102:3615-3620). A mouse model of hemophilia B also can be used totest FVII polypeptides (Margaritis et al. (2004) J Clin Invest113:1025-1031). Non-mouse models of bleeding disorders also exist. FVIIpolypeptide activity can be assessed in rats with warfarin-inducedbleeding or melagatran-induced bleeding (Diness et al. (1992) Thromb Res67:233-241, Elg et al. (2001) Thromb Res 101:145-157), and rabbits withheparin-induced bleeding (Chan et al. (2003) J Thromb Haemost1:760-765). Inbred hemophilia A, hemophilia B and von Willebrand diseasedogs that display severe bleeding also can be used in non-human animalstudies with FVII polypeptides (Brinkhous et al. (1989) PNAS86:1382-1386). The activity of FVII polypeptides also can be assessed ina rabbit model of bleeding in which thrombocytopenia is induced by acombination of gamma-irradiation and the use of platelet antibodies(Tranholm et al. (2003) Thromb Res 109:217-223).

In addition to animals with generalized bleeding disorders, injury andtrauma models also can be used to evaluate the activity of FVIIpolypeptides, and their safety and efficacy as a coagulant therapeutic.Non-limiting examples of such models include a rabbit coronary stenosismodel (Fatorutto et al. (2004) Can J Anaesth 51:672-679), a grade Vliver injury model in pigs (Lynn et al. (2002) J Trauma 52:703-707), agrade V liver injury model in pigs (Martinowitz et al. (2001) J Trauma50:721-729) and a pig aortotomy model (Sondeen et al. (2004) Shock22:163-168).

3. Clinical Assays

Many assays are available to assess activity of FVII for clinical use.Such assays can include assessment of coagulation, protein stability andhalf-life in vivo, and phenotypic assays. Phenotypic assays and assaysto assess the therapeutic effect of FVII treatment include assessment ofblood levels of FVII (e.g. measurement of serum FVII prior toadministration and time-points following administrations including,after the first administration, immediately after last administration,and time-points in between, correcting for the body mass index (BMI)),assessment of blood coagulation in vitro using the methods describedabove following treatment with FVII (e.g. PT assay), and phenotypicresponse to FVII treatment including amelioration of symptoms over timecompared to subjects treated with an unmodified and/or wild type FVII orplacebo. Patients treated with FVII polypeptides can be monitored forblood loss, transfusion requirement, and hemoglobin. Patients can bemonitored regularly over a period of time for routine or repeatedadministrations, or following administration in response to acuteevents, such as hemorrhage, trauma, or surgical procedures.

G. Formulation and Administration

Compositions for use in treatment of bleeding disorders are providedherein. Such compositions contain a therapeutically effective amount ofa factor VII polypeptide as described herein. Effective concentrationsof FVII polypeptides or pharmaceutically acceptable derivatives thereofare mixed with a suitable pharmaceutical carrier or vehicle forsystemic, topical or local administration. Compounds are included in anamount effective for treating the selected disorder. The concentrationof active compound in the composition will depend on absorption,inactivation, excretion rates of the active compound, the dosageschedule, and amount administered as well as other factors known tothose of skill in the art.

Pharmaceutical carriers or vehicles suitable for administration of thecompounds provided herein include any such carriers known to thoseskilled in the art to be suitable for the particular mode ofadministration. Pharmaceutical compositions that include atherapeutically effective amount of a FVII polypeptide described hereinalso can be provided as a lyophilized powder that is reconstituted, suchas with sterile water, immediately prior to administration.

1. Formulations

Pharmaceutical compositions containing a modified FVII can be formulatedin any conventional manner by mixing a selected amount of thepolypeptide with one or more physiologically acceptable carriers orexcipients. Selection of the carrier or excipient is within the skill ofthe administering profession and can depend upon a number of parameters.These include, for example, the mode of administration (i.e., systemic,oral, nasal, pulmonary, local, topical, or any other mode) and disordertreated. The pharmaceutical compositions provided herein can beformulated for single dosage (direct) administration or for dilution orother modification. The concentrations of the compounds in theformulations are effective for delivery of an amount, uponadministration, that is effective for the intended treatment. Typically,the compositions are formulated for single dosage administration. Toformulate a composition, the weight fraction of a compound or mixturethereof is dissolved, suspended, dispersed, or otherwise mixed in aselected vehicle at an effective concentration such that the treatedcondition is relieved or ameliorated.

The modified FVII polypeptides provided herein can be formulated foradministration to a subject as a two-chain FVIIa protein. The modifiedFVII polypeptides can be activated by any method known in the art priorto formulation. For example, FVII can undergo autoactivation duringpurification by ion exchange chromatography (Jurlander et al. (2001)Semin Thromb Hemost 27:373-384). The modified FVII polypeptides also canbe activated by incubation with FXa immobilized on beads (Kemball-Cooket al. (1998) J Biol Chem 273:8516-8521), or any other methods known inthe art (see also Example 2 below). The inclusion of calcium in theseprocesses ensures full activation and correct folding of the modifiedFVIIa protein. The modified FVII polypeptides provided herein also canbe formulated for administration as a single chain protein. Thesingle-chain FVII polypeptides can be purified in such a way as toprevent cleavage (see, e.g., U.S. Pat. No. 6,677,440). The modified FVIIpolypeptides provided herein can be formulated such that thesingle-chain and two-chain forms are contained in the pharmaceuticalcomposition, in any ratio by appropriate selection of the medium toeliminate or control autoactivation.

The compound can be suspended in micronized or other suitable form orcan be derivatized to produce a more soluble active product. The form ofthe resulting mixture depends upon a number of factors, including theintended mode of administration and the solubility of the compound inthe selected carrier or vehicle. The resulting mixtures are solutions,suspensions, emulsions and other such mixtures, and can be formulated asan non-aqueous or aqueous mixture, creams, gels, ointments, emulsions,solutions, elixirs, lotions, suspensions, tinctures, pastes, foams,aerosols, irrigations, sprays, suppositories, bandages, or any otherformulation suitable for systemic, topical or local administration. Forlocal internal administration, such as, intramuscular, parenteral orintra-articular administration, the polypeptides can be formulated as asolution suspension in an aqueous-based medium, such as isotonicallybuffered saline or are combined with a biocompatible support orbioadhesive intended for internal administration. The effectiveconcentration is sufficient for ameliorating the targeted condition andcan be empirically determined. To formulate a composition, the weightfraction of compound is dissolved, suspended, dispersed, or otherwisemixed in a selected vehicle at an effective concentration such that thetargeted condition is relieved or ameliorated.

Generally, pharmaceutically acceptable compositions are prepared in viewof approvals for a regulatory agency or other prepared in accordancewith generally recognized pharmacopeia for use in animals and in humans.Pharmaceutical compositions can include carriers such as a diluent,adjuvant, excipient, or vehicle with which an isoform is administered.Such pharmaceutical carriers can be sterile liquids, such as water andoils, including those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, and sesame oil.Water is a typical carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions also can be employed as liquid carriers, particularlyfor injectable solutions. Compositions can contain along with an activeingredient: a diluent such as lactose, sucrose, dicalcium phosphate, orcarboxymethylcellulose; a lubricant, such as magnesium stearate, calciumstearate and talc; and a binder such as starch, natural gums, such asgum acaciagelatin, glucose, molasses, polyinylpyrrolidine, cellulosesand derivatives thereof, povidone, crospovidones and other such bindersknown to those of skill in the art. Suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, andethanol. A composition, if desired, also can contain minor amounts ofwetting or emulsifying agents, or pH buffering agents, for example,acetate, sodium citrate, cyclodextrine derivatives, sorbitanmonolaurate, triethanolamine sodium acetate, triethanolamine oleate, andother such agents. These compositions can take the form of solutions,suspensions, emulsion, tablets, pills, capsules, powders, and sustainedrelease formulations. Capsules and cartridges of e.g., gelatin for usein an inhaler or insufflator can be formulated containing a powder mixof a therapeutic compound and a suitable powder base such as lactose orstarch. A composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, and other such agents. Preparations for oraladministration also can be suitably formulated with protease inhibitors,such as a Bowman-Birk inhibitor, a conjugated Bowman-Birk inhibitor,aprotinin and camostat. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin. Suchcompositions will contain a therapeutically effective amount of thecompound, generally in purified form, together with a suitable amount ofcarrier so as to provide the form for proper administration to a subjector patient.

The formulation should suit the mode of administration. For example, themodified FVII can be formulated for parenteral administration byinjection (e.g., by bolus injection or continuous infusion). Theinjectable compositions can take such forms as suspensions, solutions oremulsions in oily or aqueous vehicles. The sterile injectablepreparation also can be a sterile injectable solution or suspension in anon-toxic parenterally-acceptable diluent or solvent, for example, as asolution in 1,4-butanediol. Sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil can be employed, including, but not limited to, syntheticmono- or diglycerides, fatty acids (including oleic acid), naturallyoccurring vegetable oils like sesame oil, coconut oil, peanut oil,cottonseed oil, and other oils, or synthetic fatty vehicles like ethyloleate. Buffers, preservatives, antioxidants, and the suitableingredients, can be incorporated as required, or, alternatively, cancomprise the formulation.

The polypeptides can be formulated as the sole pharmaceutically activeingredient in the composition or can be combined with other activeingredients. The polypeptides can be targeted for delivery, such as byconjugation to a targeting agent, such as an antibody. Liposomalsuspensions, including tissue-targeted liposomes, also can be suitableas pharmaceutically acceptable carriers. These can be prepared accordingto methods known to those skilled in the art. For example, liposomeformulations can be prepared as described in U.S. Pat. No. 4,522,811.Liposomal delivery also can include slow release formulations, includingpharmaceutical matrices such as collagen gels and liposomes modifiedwith fibronectin (see, for example, Weiner et al. (1985) J Pharm Sci.74(9): 922-5). The compositions provided herein further can contain oneor more adjuvants that facilitate delivery, such as, but are not limitedto, inert carriers, or colloidal dispersion systems. Representative andnon-limiting examples of such inert carriers can be selected from water,isopropyl alcohol, gaseous fluorocarbons, ethyl alcohol, polyvinylpyrrolidone, propylene glycol, a gel-producing material, stearylalcohol, stearic acid, spermaceti, sorbitan monooleate, methylcellulose,as well as suitable combinations of two or more thereof. The activecompound is included in the pharmaceutically acceptable carrier in anamount sufficient to exert a therapeutically useful effect in theabsence of undesirable side effects on the subject treated. Thetherapeutically effective concentration can be determined empirically bytesting the compounds in known in vitro and in vivo systems, such as theassays provided herein.

a. Dosages

The precise amount or dose of the therapeutic agent administered dependson the particular FVII polypeptide, the route of administration, andother considerations, such as the severity of the disease and the weightand general state of the subject. Local administration of thetherapeutic agent will typically require a smaller dosage than any modeof systemic administration, although the local concentration of thetherapeutic agent can, in some cases, be higher following localadministration than can be achieved with safety upon systemicadministration. If necessary, a particular dosage and duration andtreatment protocol can be empirically determined or extrapolated. Forexample, exemplary doses of recombinant and native FVII polypeptides canbe used as a starting point to determine appropriate dosages. Forexample, a recombinant FVII (rFVIIa) polypeptide that has been activatedto rFVIIa, Novoseven®, has been administered to patients with hemophiliaA or hemophilia B, who are experiencing a bleeding episode, at a dosageof 90 μg/kg by bolus infusion over 2 to 5 minutes, achieving aneffective circulating level of at least 2 μg/ml. The dose is repeatedevery 2 hours until hemostasis is achieved. The modified FVIIpolypeptides provided herein can be effective at reduced dosage amountsand/or frequencies compared to such a recombinant FVII. For example, atthe modified FVII polypeptides provided herein can be administered at adosage of 80 μg/kg, 70 μg/kg, 60 μg/kg, 50 μg/kg, 40 μg/kg, 30 μg/kg, 20μg/kg, 15 μg/kg or less. In some embodiments, the dosages can be higher,such as 100 μg/kg, 110 μg/kg, 120 μg/kg, or higher. The duration oftreatment and the interval between injections will vary with theseverity of the bleed and the response of the patient to the treatment,and can be adjusted accordingly. Factors such as the level of activityand half-life of the modified FVII in comparison to the unmodified FVIIcan be taken into account when making dosage determinations. Particulardosages and regimens can be empirically determined.

In another example, a recombinant FVII (rFVIIa) polypeptide that hasbeen activated to rFVIIa, Novoseven®, has been administered to patientswith congenital FVII deficiency who are experiencing a bleeding episode,at a dosage of 15-30 μg/kg by bolus infusion over 2 to 5 minutes. Thedose is repeated every 4-6 hours until hemostasis is achieved. Themodified FVII polypeptides provided herein can be effective at reduceddosage amounts and/or frequencies compared to such a recombinant FVII.For example, the modified FVII polypeptides provided herein can beadministered at a dosage of 20 μg/kg, 15 μg/kg, 10 μg/kg, 5 μg/kg, 3μg/kg or less. In some examples, the dosages can be higher, such as 35μg/kg, 40 μg/kg, 45 μg/kg, or higher. The duration of treatment and theinterval between injections will vary with the severity of the bleed andthe response of the patient to the treatment, and can be adjustedaccordingly. Factors such as the level of activity and half-life of themodified FVII in comparison to the unmodified FVII can be used in makingdosage determinations. For example, a modified FVII polypeptide thatexhibits a longer half-life than an unmodified FVII polypeptide can beadministered at lower doses and/or less frequently than the unmodifiedFVII polypeptide. Similarly, the dosages required for therapeutic effectusing a modified FVII polypeptide that displays increased coagulantactivity compared with an unmodified FVII polypeptide can be reduced infrequency and amount. Particular dosages and regimens can be empiricallydetermined by one of skill in the art.

b. Dosage Forms

Pharmaceutical therapeutically active compounds and derivatives thereofare typically formulated and administered in unit dosage forms ormultiple dosage forms. Formulations can be provided for administrationto humans and animals in dosage forms that include, but are not limitedto, tablets, capsules, pills, powders, granules, sterile parenteralsolutions or suspensions, oral solutions or suspensions, and oil wateremulsions containing suitable quantities of the compounds orpharmaceutically acceptable derivatives thereof. Each unit dose containsa predetermined quantity of therapeutically active compound sufficientto produce the desired therapeutic effect, in association with therequired pharmaceutical carrier, vehicle or diluent. Examples of unitdose forms include ampoules and syringes and individually packagedtablets or capsules. In some examples, the unit dose is provided as alyophilized powder that is reconstituted prior to administration. Forexample, a FVII polypeptide can be provided as lyophilized powder thatis reconstituted with a suitable solution to generate a single dosesolution for injection. In some embodiments, the lyophilized powder cancontain the FVII polypeptide and additional components, such as salts,such that reconstitution with sterile distilled water results in a FVIIpolypeptide in a buffered or saline solution. Unit dose forms can beadministered in fractions or multiples thereof. A multiple dose form isa plurality of identical unit dosage forms packaged in a singlecontainer to be administered in segregated unit dose form. Examples ofmultiple dose forms include vials, bottles of tablets or capsules orbottles of pints or gallons. Hence, multiple dose form is a multiple ofunit doses that are not segregated in packaging.

2. Administration of Modified FVII Polypeptides

The FVII polypeptides provided herein (i.e. active compounds) can beadministered in vitro, ex vivo, or in vivo by contacting a mixture, suchas a body fluid or other tissue sample, with a FVII polypeptide. Forexample, when administering a compound ex vivo, a body fluid or tissuesample from a subject can be contacted with the FVII polypeptides thatare coated on a tube or filter, such as for example, a tube or filter ina bypass machine. When administering in vivo, the active compounds canbe administered by any appropriate route, for example, orally, nasally,pulmonary, parenterally, intravenously, intradermally, subcutaneously,intraarticularly, intracisternally, intraocularly, intraventricularly,intrathecally, intramuscularly, intraperitoneally, intratracheally ortopically, as well as by any combination of any two or more thereof, inliquid, semi-liquid or solid form and are formulated in a mannersuitable for each route of administration. The modified FVIIpolypeptides can be administered once or more than once, such as twice,three times, four times, or any number of times that are required toachieve a therapeutic effect. Multiple administrations can be effectedvia any route or combination of routes, and can be administered hourly,every 2 hours, every three hours, every four hours or more.

The most suitable route for administration will vary depending upon thedisease state to be treated, for example the location of the bleedingdisorder. Generally, the FVII polypeptides will be administered byintravenous bolus injection, with an administration (infusing) time ofapproximately 2-5 minutes. In other examples, desirable blood levels ofFVII can be maintained by a continuous infusion of the active agent asascertained by plasma levels. It should be noted that the attendingphysician would know how to and when to terminate, interrupt or adjusttherapy to lower dosage due to toxicity, or bone marrow, liver or kidneydysfunctions. Conversely, the attending physician would also know how toand when to adjust treatment to higher levels if the clinical responseis not adequate (precluding toxic side effects). In other examples, thelocation of the bleeding disorder might indicate that the FVIIformulation is administered via alternative routes. For example, localadministration, including administration into the brain (e.g.,intraventricularly) might be performed when the patient is experiencingbleeding in this region. Similarly, for treatment of bleeding in thejoints, local administration by injection of the therapeutic agent intothe joint (i.e., intraarticularly, intravenous or subcutaneous means)can be employed. In other examples, topical administration of thetherapeutic agent to the skin, for example formulated as a cream, gel,or ointment, or administration to the lungs by inhalation orintratracheally, might be appropriate when the bleeding is localized tothese areas.

The instances where the modified FVII polypeptides are be formulated asa depot preparation, the long-acting formulations can be administered byimplantation (for example, subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the therapeutic compoundscan be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The compositions, if desired, can be presented in a package, in a kit ordispenser device, that can contain one or more unit dosage formscontaining the active ingredient. The package, for example, containsmetal or plastic foil, such as a blister pack. The pack or dispenserdevice can be accompanied by instructions for administration. Thecompositions containing the active agents can be packaged as articles ofmanufacture containing packaging material, an agent provided herein, anda label that indicates the disorder for which the agent is provided.

3. Administration of Nucleic Acids Encoding Modified FVII Polypeptides(Gene Therapy)

Also provided are compositions of nucleic acid molecules encoding themodified FVII polypeptides and expression vectors encoding them that aresuitable for gene therapy. Rather than deliver the protein, nucleic acidcan be administered in vivo, such as systemically or by other route, orex vivo, such as by removal of cells, including lymphocytes,introduction of the nucleic therein, and reintroduction into the host ora compatible recipient.

Modified FVII polypeptides can be delivered to cells and tissues byexpression of nucleic acid molecules. Modified FVII polypeptides can beadministered as nucleic acid molecules encoding modified FVIIpolypeptides, including ex vivo techniques and direct in vivoexpression. Nucleic acids can be delivered to cells and tissues by anymethod known to those of skill in the art. The isolated nucleic acidsequences can be incorporated into vectors for further manipulation. Asused herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous DNA into cells for either expression orreplication thereof. Selection and use of such vehicles are well withinthe skill of the artisan.

Methods for administering modified FVII polypeptides by expression ofencoding nucleic acid molecules include administration of recombinantvectors. The vector can be designed to remain episomal, such as byinclusion of an origin of replication or can be designed to integrateinto a chromosome in the cell. Modified FVII polypeptides also can beused in ex vivo gene expression therapy using non-viral vectors. Forexample, cells can be engineered to express a modified FVII polypeptide,such as by integrating a modified FVII polypeptide encoding-nucleic acidinto a genomic location, either operatively linked to regulatorysequences or such that it is placed operatively linked to regulatorysequences in a genomic location. Such cells then can be administeredlocally or systemically to a subject, such as a patient in need oftreatment.

Viral vectors, include, for example adenoviruses, adeno-associatedviruses (AAV), poxviruses, herpes viruses, retroviruses and othersdesigned for gene therapy can be employed. The vectors can remainepisomal or can integrate into chromosomes of the treated subject. Amodified FVII polypeptide can be expressed by a virus, which isadministered to a subject in need of treatment. Viral vectors suitablefor gene therapy include adenovirus, adeno-associated virus (AAV),retroviruses, lentiviruses, vaccinia viruses and others noted above. Forexample, adenovirus expression technology is well-known in the art andadenovirus production and administration methods also are well known.Adenovirus serotypes are available, for example, from the American TypeCulture Collection (ATCC, Rockville, Md.). Adenovirus can be used exvivo, for example, cells are isolated from a patient in need oftreatment, and transduced with a modified FVII polypeptide-expressingadenovirus vector. After a suitable culturing period, the transducedcells are administered to a subject, locally and/or systemically.Alternatively, modified FVII polypeptide-expressing adenovirus particlesare isolated and formulated in a pharmaceutically-acceptable carrier fordelivery of a therapeutically effective amount to prevent, treat orameliorate a disease or condition of a subject. Typically, adenovirusparticles are delivered at a dose ranging from 1 particle to 10¹⁴particles per kilogram subject weight, generally between 10⁶ or 10⁸particles to 10¹² particles per kilogram subject weight. In somesituations it is desirable to provide a nucleic acid source with anagent that targets cells, such as an antibody specific for a cellsurface membrane protein or a target cell, or a ligand for a receptor ona target cell. FVII also can be targeted for delivery into specific celltypes. For example, adenoviral vectors encoding FVII polypeptides can beused for stable expression in nondividing cells, such as liver cells(Margaritis et al. (2004) J Clin Invest 113:1025-1031). In anotherexample, viral or nonviral vectors encoding FVII polypeptides can betransduced into isolated cells for subsequent delivery. Additional celltypes for expression and delivery of FVII might include, but are notlimited to, fibroblasts and endothelial cells.

The nucleic acid molecules can be introduced into artificial chromosomesand other non-viral vectors. Artificial chromosomes, such as ACES (see,Lindenbaum et al. (2004) Nucleic Acids Res. 32(21):e172) can beengineered to encode and express the isoform. Briefly, mammalianartificial chromosomes (MACs) provide a means to introduce largepayloads of genetic information into the cell in an autonomouslyreplicating, non-integrating format. Unique among MACs, the mammaliansatellite DNA-based Artificial Chromosome Expression (ACE) can bereproducibly generated de novo in cell lines of different species andreadily purified from the host cells' chromosomes. Purified mammalianACEs can then be re-introduced into a variety of recipient cell lineswhere they have been stably maintained for extended periods in theabsence of selective pressure using an ACE System. Using this approach,specific loading of one or two gene targets has been achieved in LMTK(−)and CHO cells.

Another method for introducing nucleic acids encoding the modified FVIIpolypeptides is a two-step gene replacement technique in yeast, startingwith a complete adenovirus genome (Ad2; Ketner et al. (1994) PNAS 91:6186-6190) cloned in a Yeast Artificial Chromosome (YAC) and a plasmidcontaining adenovirus sequences to target a specific region in the YACclone, an expression cassette for the gene of interest and a positiveand negative selectable marker. YACs are of particular interest becausethey permit incorporation of larger genes. This approach can be used forconstruction of adenovirus-based vectors bearing nucleic acids encodingany of the described modified FVII polypeptides for gene transfer tomammalian cells or whole animals.

The nucleic acids can be encapsulated in a vehicle, such as a liposome,or introduced into a cells, such as a bacterial cell, particularly anattenuated bacterium or introduced into a viral vector. For example,when liposomes are employed, proteins that bind to a cell surfacemembrane protein associated with endocytosis can be used for targetingand/or to facilitate uptake, e.g. capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life.

For ex vivo and in vivo methods, nucleic acid molecules encoding themodified FVII polypeptide is introduced into cells that are from asuitable donor or the subject to be treated. Cells into which a nucleicacid can be introduced for purposes of therapy include, for example, anydesired, available cell type appropriate for the disease or condition tobe treated, including but not limited to epithelial cells, endothelialcells, keratinocytes, fibroblasts, muscle cells, hepatocytes; bloodcells such as T lymphocytes, B lymphocytes, monocytes, macrophages,neutrophils, eosinophils, megakaryocytes, granulocytes; various stem orprogenitor cells, in particular hematopoietic stem or progenitor cells,e.g., such as stem cells obtained from bone marrow, umbilical cordblood, peripheral blood, fetal liver, and other sources thereof.

For ex vivo treatment, cells from a donor compatible with the subject tobe treated or the subject to be treated cells are removed, the nucleicacid is introduced into these isolated cells and the modified cells areadministered to the subject. Treatment includes direct administration,such as, for example, encapsulated within porous membranes, which areimplanted into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and5,283,187 each of which is herein incorporated by reference in itsentirety). Techniques suitable for the transfer of nucleic acid intomammalian cells in vitro include the use of liposomes and cationiclipids (e.g., DOTMA, DOPE and DC-Chol) electroporation, microinjection,cell fusion, DEAE-dextran, and calcium phosphate precipitation methods.Methods of DNA delivery can be used to express modified FVIIpolypeptides in vivo. Such methods include liposome delivery of nucleicacids and naked DNA delivery, including local and systemic delivery suchas using electroporation, ultrasound and calcium-phosphate delivery.Other techniques include microinjection, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer andspheroplast fusion.

In vivo expression of a modified FVII polypeptide can be linked toexpression of additional molecules. For example, expression of amodified FVII polypeptide can be linked with expression of a cytotoxicproduct such as in an engineered virus or expressed in a cytotoxicvirus. Such viruses can be targeted to a particular cell type that is atarget for a therapeutic effect. The expressed modified FVII polypeptidecan be used to enhance the cytotoxicity of the virus.

In vivo expression of a modified FVII polypeptide can includeoperatively linking a modified FVII polypeptide encoding nucleic acidmolecule to specific regulatory sequences such as a cell-specific ortissue-specific promoter. Modified FVII polypeptides also can beexpressed from vectors that specifically infect and/or replicate intarget cell types and/or tissues. Inducible promoters can be use toselectively regulate modified FVII polypeptide expression. An exemplaryregulatable expression system is the doxycycline-inducible geneexpression system, which has been used to regulate recombinant FVIIexpression (Srour et al. (2003) Thromb Haemost. 90(3): 398-405).

Nucleic acid molecules, as naked nucleic acids or in vectors, artificialchromosomes, liposomes and other vehicles can be administered to thesubject by systemic administration, topical, local and other routes ofadministration. When systemic and in vivo, the nucleic acid molecule orvehicle containing the nucleic acid molecule can be targeted to a cell.

Administration also can be direct, such as by administration of a vectoror cells that typically targets a cell or tissue. For example, tumorcells and proliferating can be targeted cells for in vivo expression ofmodified FVII polypeptides. Cells used for in vivo expression of anmodified FVII polypeptide also include cells autologous to the patient.Such cells can be removed from a patient, nucleic acids for expressionof an modified FVII polypeptide introduced, and then administered to apatient such as by injection or engraftment.

H. Therapeutic Uses

The modified FVII polypeptides provided herein can be used for treatmentof any condition for which recombinant FVII is employed. Typically, suchtreatments include those where increased coagulation, such as increasedhemostatic responses, are desired. Modified FVII polypeptides havetherapeutic activity alone or in combination with other agents. Themodified polypeptides provided herein are designed to retain therapeuticactivity but exhibit modified properties, particularly increasedresistance to AT-III and increased catalytic activity. The modifiedpolypeptides provided herein also can exhibit increased resistance toTFPI, increased resistance to the inhibitory effects of Zn²⁺, improvedpharmacokinetic properties, such as serum half-life, increased bindingand/or affinity for activated platelets, increased binding and/oraffinity for serum albumin, and/or increased binding and/or affinity forplatelet integrin α_(IIb)β₃. Such modified properties, for example, canimprove the therapeutic effectiveness of the polypeptides due toincreased coagulant activity of the modified FVII polypeptides. Thissection provides exemplary uses of and administration methods. Thesedescribed therapies are exemplary and do not limit the applications ofmodified FVII polypeptides.

The modified FVII polypeptides provided herein can be used in varioustherapeutic as well as diagnostic methods in which FVII is employed.Such methods include, but are not limited to, methods of treatment ofphysiological and medical conditions described and listed below.Modified FVII polypeptides provided herein can exhibit improvement of invivo activities and therapeutic effects compared to wild-type FVII,including lower dosage to achieve the same effect, and otherimprovements in administration and treatment such as fewer and/or lessfrequent administrations, decreased side effects and increasedtherapeutic effects. Although it is understood that the modified FVIIpolypeptides can be administered as a FVII zymogen (i.e. single chainform), typically the modified FVII polypeptides provided herein areadministered in activated two-chain form following, for example,autoactivation or activation by other coagulation factors, such asduring purification.

In particular, modified FVII polypeptides are intended for use intherapeutic methods in which FVII has been used for treatment. Suchmethods include, but are not limited to, methods of treatment ofdiseases and disorders, such as, but not limited to, blood coagulationdisorders, hematologic disorders, hemorrhagic disorders, hemophilias,such as hemophilia A, hemophilia B and factor VII deficiency, andacquired blood disorders, such as acquired factor VII deficiency causedby liver disease. Modified FVII polypeptides also can be used in thetreatment of additional bleeding diseases and disorders, such as, butnot limited to, thrombocytopenia (e.g., such as due to chemotherapeuticregimes), Von Willebrand's disease, hereditary platelet disorders (e.g.,storage pool disease such as Chediak-Higashi and Hermansky-Pudlaksyndromes, thromboxane A2 dysfunction, Glanzmann's thrombasthenia, andBemard-Soulier syndrome), hemolytic-uremic syndrome, HereditaryHemorrhagic Telangiectsasia, also known as Rendu-Osler-Weber syndrome,allergic purpura (Henoch Schonlein purpura) and disseminatedintravascular coagulation.

In some embodiments, the bleedings to be treated by FVII polypeptidesoccur in organs such as the brain, inner ear region, eyes, liver, lung,tumor tissue, gastrointestinal tract. In other embodiments, the bleedingis diffuse, such as in haemorrhagic gastritis and profuse uterinebleeding. Patients with bleeding disorders, such as for example,hemophilia A and B, often are at risk of bleeding complications duringsurgery or trauma. Such bleeding can be manifested as acutehaemarthroses (bleedings in joints), chronic hemophilic arthropathy,haematomas, (e.g., muscular, retroperitoneal, sublingual andretropharyngeal), haematuria (bleeding from the renal tract), centralnervous system bleedings, gastrointestinal bleedings (e.g., UGI bleeds)and cerebral hemorrhage, which also can be treated with modified FVIIpolypeptides. Additionally, any bleeding associated with surgery (e.g.,hepatectomy), or dental extraction can be treated with modified FVIIpolypeptides. In one embodiment, the modified FVII polypeptides can beused to treat bleeding episodes due to trauma, or surgery, or loweredcount or activity of platelets, in a subject. Exemplary methods forpatients undergoing surgery include treatments to prevent hemorrhage andtreatments before, during, or after surgeries such as, but not limitedto, heart surgery, angioplasty, lung surgery, abdominal surgery, spinalsurgery, brain surgery, vascular surgery, dental surgery, or organtransplant surgery, including transplantation of bone marrow, heart,lung, pancreas, or liver.

Treatment of diseases and conditions with modified FVII polypeptides canbe effected by any suitable route of administration using suitableformulations as described herein including, but not limited to,injection, pulmonary, oral and transdermal administration. Treatmenttypically is effected by intravenous bolus administration.

If necessary, a particular dosage and duration and treatment protocolcan be empirically determined or extrapolated. For example, exemplarydoses of recombinant and native FVII polypeptides can be used as astarting point to determine appropriate dosages. For example, arecombinant FVII (rFVIIa) polypeptide that has been activated to rFVIIa,Novoseven®, has been administered to patients with hemophilia A orhemophilia B, who are experiencing a bleeding episode, at a dosage of 90μg/kg by bolus infusion over 2 to 5 minutes, achieving an effectivecirculating level of at least 2 μg/ml, with a mean half-life of 2.7hours. The dose is repeated every 2 hours until hemostasis is achieved.Modified FVII polypeptides that are have an increased coagulantactivity, due to, for example, increased resistance to AT-III, increasedcatalytic activity, increased resistance to the inhibitory effects ofZn²⁺, increased resistance to TFPI, improved pharmacokinetic properties,such as increased serum half-life, increased binding and/or affinity foractivated platelets, increased binding and/or affinity for serumalbumin, and/or increased binding and/or affinity for platelet integrinα_(IIb)β₃, can be effective at reduced dosage amounts and/or frequenciescompared to such a recombinant FVII. Dosages for wild-type or unmodifiedFVII polypeptides can be used as guidance for determining dosages formodified FVII polypeptides. Factors such as the level of activity andhalf-life of the modified FVII in comparison to the unmodified FVII canbe used in making such determinations. Particular dosages and regimenscan be empirically determined.

Dosage levels and regimens can be determined based upon known dosagesand regimens, and, if necessary can be extrapolated based upon thechanges in properties of the modified polypeptides and/or can bedetermined empirically based on a variety of factors. Such factorsinclude body weight of the individual, general health, age, the activityof the specific compound employed, sex, diet, time of administration,rate of excretion, drug combination, the severity and course of thedisease, and the patient's disposition to the disease and the judgmentof the treating physician. The active ingredient, the polypeptide,typically is combined with a pharmaceutically effective carrier. Theamount of active ingredient that can be combined with the carriermaterials to produce a single dosage form or multi-dosage form can varydepending upon the host treated and the particular mode ofadministration.

The effect of the FVII polypeptides on the clotting time of blood can bemonitored using any of the clotting tests known in the art including,but not limited to, whole blood prothrombin time (PT), the activatedpartial thromboplastin time (aPTT), the activated clotting time (ACT),the recalcified activated clotting time, or the Lee-White Clotting time.

Upon improvement of a patient's condition, a maintenance dose of acompound or compositions can be administered, if necessary; and thedosage, the dosage form, or frequency of administration, or acombination thereof can be modified. In some cases, a subject canrequire intermittent treatment on a long-term basis upon any recurrenceof disease symptoms or based upon scheduled dosages. In other cases,additional administrations can be required in response to acute eventssuch as hemorrhage, trauma, or surgical procedures.

The following are some exemplary conditions for which FVII (administeredas FVIIa) has been used as a treatment agent alone or in combinationwith other agents.

1. Congenital Bleeding Disorders

a. Hemophilia

Congenital hemophilia is a recessive blood disorder in which there aredecreased levels of coagulation factors in the plasma, leading todisruption of the coagulation cascade and increased blot clotting time.Hemophilia A, which accounts for approximately 85% of all cases ofhemophilia, results from mutations(s) in the factor VIII gene on the Xchromosome, leading to a deficiency or dysfunction of the FVIII protein.Hemophilia B is caused by a deficiency or dysfunction of the coagulationfactor, FIX, generally resulting from point mutations or deletions inthe FIX gene on X chromosome. The worldwide incidence of hemophilia A isapproximately 1 case per 5000 male individuals, and 1 case per 25000males for hemophilia B. Hemophilia A and B are further classified asmild, moderate, or severe. A plasma level with 5%-25% of normallyfunctioning factor VIII or IX is classified as mild, 1%-5% is moderate,and less that 1% is severe. Hemophilia C, often referred to as FIXdeficiency, is a relatively mild and rare disease, affecting about 1 in100000 people in an autosomal recessive manner.

Hemophilia A and B manifests clinically in many ways. Minor cuts andabrasions will not result in excessive bleeding, but traumas andsurgeries will. The patient also will have numerous joint and musclebleeds and easy bruising. Hemarthrosis or bleeding into the joints isone of the major complications in hemophilia, and can occurspontaneously or in response to trauma. The hinge joints, such as theknee, elbow and ankle, are affected most frequently. The hip andshoulder are affected much less frequently as the ball and socket jointhave more musculature surrounding them, thus protecting them more frominjury. The bleeding can cause severe acute pain, restrict movement, andlead to secondary complications including synovial hypertrophy.Furthermore, the recurring bleeding in the joints can cause chronicsynovitis, which can cause joint damage, destroying synovium, cartilage,and bone. Life-threatening hemorrhages, such as intracranial hemorrhageand bleeding in the central nervous system, also afflicts hemophilicsubjects. Intracranial bleeding occurs in approximately 10% of patientswith sever hemophilia, resulting in a 30% mortality rate. In contrast,Hemophilia C is more mild. Spontaneous bleeds are rarely seen, andbleeding into joints, soft tissues and muscles also is uncommon.Bleeding is generally treated with transfusion of fresh frozen plasma(FFP), FXI replacement therapy, or, for topical treatment, suchtreatment of external wounds or dental extractions, fibrin glue.

The most common treatment for hemophilia A or B is replacement therapy,in which the patient is administered FVIII or FIX. The formulations areavailable commercially as plasma-derived or recombinant products, withrecombinant proteins now being the treatment of choice in previouslyuntreated patients. While these therapies can be very successful,complications arise if the patient develops inhibitors to the newlyadministered factor VIII or factor IX. Inhibitors are IgG antibodies,mostly of the IgG4 subclass, that react with FVIII or FIX and interferewith pro-coagulant function. Inhibitors affect about 1 in 5 patientswith severe hemophilia A. Most subjects develop these inhibitors soonafter administration of the first infusions of factor VIII, which isoften in early childhood, although subjects develop them later in life.Inhibitors also affect about 1 in 15 people with mild or moderatehemophilia A. These inhibitors usually develop during adulthood and notonly destroy administered exogenous FVIII, but also destroy endogenousFVIII. As a result, mild and moderate hemophiliacs become severe.Clinically, hemophilia A patients with inhibitors are classified intohigh and low responders according to the strength of the anamnesticresponse they experience when they are re-exposed to FVIII. Inhibitorsaffect about 1 in 100 patients with hemophilia B. In most cases, theinhibitors develop after the first infusions of therapeutic factor IXand can be accompanied by allergic reactions.

The modified FVII polypeptides presented herein can be used to treatpatients with hemophilia, particularly hemophilia patients withinhibitors. A recombinant FVIIa product (NovoSeven, Novo Nordisk) hasbeen approved and licensed for the treatment of bleeding episodes inhemophilia A or B patients with inhibitors to FVIII or FIX and for theprevention of bleeding in surgical interventions or invasive proceduresin hemophilia A or B patients with inhibitors to FVIII or FIX. Treatmentwith rFVIIa enhances thrombin generation while bypassing the requirementfor FVIIIa and/or FIXa. Coagulation is initiated at the site of injuryby the interaction of rFVIIa with TF, resulting in initial FXactivation, thrombin generation, and activation of platelets. Completecoagulation by rFVIIa is can be effected by the TF-dependent andTF-independent mechanisms, where some of the thrombin generated canresult from the direct activation of FX on activated platelets by rFVIIaalone, which itself binds activated platelets through low affinityinteractions with the phospholipid membranes.

The modified FVII polypeptides provided herein can be used in therapiesfor hemophilia, including the treatment of bleeding episodes and theprevention of bleeding in surgical interventions or invasive procedures.The modified FVII polypeptides herein can provide increased resistanceto AT-III, increased catalytic activity, increased resistance to theinhibitory effects of Zn²⁺, increased resistance to TFPI, improvedpharmacokinetic properties, such as serum half-life, increased bindingand/or affinity for activated platelets, increased binding and/oraffinity for serum albumin, and/or increased binding and/or affinity forplatelet integrin α_(IIb)β₃. The FVII polypeptides can therefore displayhigher coagulant activity in a TF-dependent manner (such as throughincreased resistance to TFPI), and/or a TF-independent manner (such asthrough increased binding and/or affinity for activated platelets).Thus, the modified FVII polypeptides can be used to deliver more activetherapies for hemophilia. Examples of therapeutic improvements usingmodified FVII polypeptides include for example, but are not limited to,lower dosages, fewer and/or less frequent administrations, decreasedside effects, and increased therapeutic effects.

The modified FVII polypeptides typically are administered as activatedFVII (FVIIa) polypeptides. Modified FVII polypeptides can be tested fortherapeutic effectiveness, for example, by using animal models. Forexample antibody-induced hemophilic mice, or any other known diseasemodel for hemophilia, can be treated with modified FVII polypeptides.Progression of disease symptoms and phenotypes is monitored to assessthe effects of the modified FVII polypeptides. Modified FVIIpolypeptides also can be administered to subjects such as in clinicaltrials to assess in vivo effectiveness in comparison to placebo controlsand/or controls using unmodified FVII.

b. FVII Deficiency

Factor VII deficiency is an autosomal recessive bleeding disorder thataffects approximately 1 in 500000 people. FVII deficiency can beclinically mild, moderate or severe, with mild to moderate deficiencycharacterized by increased bleeding after surgery and trauma. Patientswith severe FVII deficiency (less than 1% FVII activity) experiencesimilar symptoms to hemophilia. For example, FVII-deficient subjects areprone to joint bleeds joint bleeds, spontaneous nosebleeds,gastrointestinal bleeding, urinary tract bleeding. Intracerebralhemorrhaging and muscle bleeds have also been reported, while women canexperience severe menorrhagia (heavy menstrual bleeding). Treatment canbe effected by replacement therapy. A recombinant FVIIa product(NovoSeven®, Novo Nordisk) has been approved and licensed for thetreatment of bleeding episodes in patients with congenital FVIIdeficiency and for the prevention of bleeding in surgical interventionsor invasive procedures in patients with congenital FVII deficiency.Hence, the modified FVII polypeptides herein can be similarly used. Themodified FVII polypeptides provided herein can be used in the treatmentof bleeding episodes and the prevention of bleeding in surgicalinterventions or invasive procedures in FVII-deficient patients. Forexample, a neonatal patient presenting with severe FVII deficiency withintracranial hemorrhaging can be administered modified FVII polypeptidesby intravenous bolus to effect coagulation and maintain hemostasis.Generally the modified FVII polypeptides are administered as activatedFVII (FVIIa) polypeptides.

c. Others

Other bleeding disorders can be treated with the FVII polypeptidesprovided herein to promote coagulation. Congenital deficiencies offactors V and X also present with increased blood clotting times and canpotentially be treated with administration of therapeutic doses of FVII.For example, a patient with factor X deficiency can be administeredrFVIIa to control bleeding associated with splenectomy (Boggio et al.(2001) Br J Haematol 112:1074-1075). Spontaneous and surgery associatedbleeding episodes associated with von Willebrand disease (vWD) also canbe treated using the modified FVII polypeptides provided herein. VWD isa bleeding disorder caused by a defect or deficiency of the bloodclotting protein, von Willebrand Factor (vWF), and is estimated to occurin 1% to 2% of the population. Subjects with vWD bruise easily, haverecurrent nosebleeds, bleed after tooth extraction, tonsillectomy orother surgery, and women patients can have increased menstrual bleeding.Modified FVII polypeptides can be used to ameliorate spontaneous andsurgery-associated bleeding in vWD patients (von Depka et al. (2006)Blood Coagul Fibrin 17:311-316). Other platelet-related bleedingdisorders, such as for example, Glanzmann's thrombasthenia andHermansky-Pudlak syndrome also are associated with reduced endogenousclotting activity. Excess spontaneous or surgery-associated bleeding inpatients with platelet related bleeding disorders also can be controlledby therapeutic doses of the modified FVII polypeptides. For example, apatient with Glanzmann's thrombasthenia undergoing surgery can betreated before, during and/or after surgery with the modified FVIIpolypeptides to prevent major blood loss (van Buuren et al. (2002) DigDis Sci 47:2134-2136). Generally, the modified FVII polypeptides areadministered as activated FVII (FVIIa) polypeptides.

2. Acquired Bleeding Disorders

a. Chemotherapy-Acquired Thrombocytopenia

Bleeding disorders also can be acquired, rather than congenital. Forexample, chemotherapy treatment, such as for leukemia and other cancers,can result in thrombocytopenia. This is likely due to a loss of plateletproduction in the bone marrow of patients receiving chemotherapy, andtypically occurs 6-10 days after medication. Treatment of the acquiredthrombocytopenia is usually by platelet, red blood cell or plasmatransfusion, which serves to prevent any abnormal spontaneous bleedingthat can result from platelet deficiency. Bleeding in patients withchemotherapy-induced thrombocytopenia, or any other acquired orcongenital thrombocytopenia, also can be controlled by administration oftherapeutic amounts of the modified FVII polypeptides provided herein.For example, a thrombocytopenic patient with uncontrolled bleeding, suchas in the gastrointestinal tract, can be administered an intravenousbolus injection of a therapeutic amount of FVII polypeptide to stophemorrhaging (Gerotziafas et al. (2002) Am J Hematol 69:219-222).Generally, the modified FVII polypeptides are administered as activatedFVII (FVIIa) polypeptides.

b. Other Coagulopathies

Other acquired coagulopathies can be treated using the modified FVIIpolypeptides presented herein. Coagulopathy can result from conditionsincluding, but not limited to, fulminant hepatic failure (FHF; such ascaused by hepatoxic drugs, toxins, metabolic diseases, infectiousdiseases and ischemia), other liver disease, including cirrhosis anddisease associated with Wilson's disease, vitamin K deficiency (such ascaused by antibiotic treatment or diet), hemolytic uremic syndrome,thrombotic thrombocytopenia (TTC) and disseminated intravascularcoagulopathy (DIC). Conventional treatment is generally by transfusionwith plasma, red blood cells (RBC), or platelets, but can beunsuccessful. In one embodiment, the modified FVII polypeptides can beadministered to a patient with FHF undergoing invasive procedures toprevent bleeding. Conventional treatment with fresh frozen plasma (FFP)often is unsuccessful and can require large quantities of plasma,producing volume overload and anasarca (a generalized infiltration ofedema fluid into subcutaneous connective tissue). Treatment withtherapeutic amounts of modified FVII polypeptides by intravenous bolusduring, before and/or after invasive surgery, such as for example, liverbiopsy or liver transplantation, can prevent bleeding and establishhemostasis in FHF patients. The patient can be monitored by PT of theblood to determine the efficacy of treatment (Shami et al. (2003) LiverTranspl 9:138-143). In another embodiment, FVII can be administered to apatient with severe bleeding associated with coagulopathy, such as forexample, severe post-cesarean intra-abdominal bleeding associated withliver dysfunction and DIC, that did not respond to conventionaltransfusions infusions (Moscardo et al. (2001) Br J Haematol113:174-176). Further, the modified FVII polypeptides can be used totreat coagulopathy in neonatal and pediatric patients. In a particularembodiment, the neonatal and pediatric patients do not respond toconventional treatment, such as RBC and platelet infusion. For example,neonates with severe pulmonary hemorrhaging associated with increasedPTs who do not respond to RBC and platelet transfusion can beadministered modified FVII polypeptides to decrease PT and establishhemostasis (Olomu et al. (2002) J Perinatol 22:672-674). The modifiedFVII polypeptides provided herein exhibit enhanced coagulation activitycompared with unmodified FVII polypeptides, and can therefore beadministered, for example, at lower doses, less frequently, and withfewer adverse reactions. Generally the modified FVII polypeptides areadministered as activated FVII (FVIIa) polypeptides.

c. Transplant-Acquired Bleeding

Severe bleeding following bone marrow transplant (BMT) and stem celltransplant (SCT) is a relatively common and life-threateningcomplication associated with these procedures, due to the reduction ofplatelets. For example, diffuse alveolar hemorrhage (DAH) is a pulmonarycomplication of BMT with an estimated incidence of 1-21% in thetransplant population, and a mortality rate of 60-100%. Conventionaltreatment of such bleeding episodes includes corticosteroid treatmentand transfusion with plasma, platelets and/or RBC, although these arelargely unsuccessful with an overall mortality rate of approximately 50%(Hicks et al. (2002) Bone Marrow Transpl 30:975-978). Administration ofFVII by intravenous bolus, with or without concurrent treatment withcorticosterioids and/or platelet infusion, can be performed to treat DAHand establish hemostasis (Hicks et al. (2002) Bone Marrow Transpl30:975-978). The modified FVII polypeptides provided herein exhibitenhanced coagulation activity compared with unmodified FVIIpolypeptides, and might therefore be administered, for example, at lowerdoses, less frequently, over a shorter treatment duration, and withfewer adverse reactions for the same biological activity and efficacy.Generally the modified FVII polypeptides are administered as activatedFVII (FVIIa) polypeptides.

d. Anticoagulant Therapy-Induced Bleeding

Patients undergoing anticoagulant therapies for the treatment ofconditions, such as thromboembolism, can exhibit bleeding episodes uponacute administration of anticoagulants, such as warfarin, heparin andfondaparinux, or develop hemorrhagic disorders as a result long termusage of such therapies. Treatments for bleeding episodes typicallyinclude administration of procoagulants, such as vitamin K, plasma,exogenous FIX, and protamines to neutralize heparin. Administration ofexogenous FVII also can be performed to neutralize the effect of theanti-coagulants, increase PT, aPTT, and/or other markers of coagulationand establish hemostasis (Deveras et al. (2002) Ann Inten Med137:884-888). The modified FVII polypeptides provided herein can be usedin treatments to control bleeding episodes in patients with acquiredbleeding disorders due to anticoagulant treatments. Generally themodified FVII polypeptides are administered as activated FVII (FVIIa)polypeptides.

e. Acquired Hemophilia

Factor VIII inhibitors can develop spontaneously in otherwise healthyindividuals, resulting in a condition known as “acquired hemophilia”.Acquired hemophilia is a rare condition, with a yearly incidence of0.2-1.0 per million population. The autoantibodies are mainly IgG4antibodies, which, when bound to FVIII, inhibit FVIII activity byinterfering with thrombin cleavage, von Willebrand factor interactionand/or phospholipid binding. This results in life-threatening hemorrhagein approximately 87% of affected patients. Common sites of bleeding areskin, mucosa, muscles and retroperitoneum, in contrast to patients withhereditary hemophilia who bleed predominantly in joints and muscles.Acquired hemophilia can be treated with an activated prothrombin complexconcentrate or recombinant activated factor VII (NovoSeven®, NovoNordisk) to control bleeding episodes. The modified FVII polypeptidesprovided herein exhibit enhanced coagulation activity compared withunmodified FVII polypeptides, and can therefore be administered, forexample, at lower doses, less frequently, over a shorter treatmentduration, and with fewer adverse reactions for the same biologicalactivity and efficacy. Generally the modified FVII polypeptides areadministered as activated FVII (FVIIa) polypeptides.

3. Trauma and Surgical Bleeding

FVII polypeptides can be used as therapy to treat bleeding associatedwith perioperative and traumatic blood loss in subjects with normalcoagulation systems. For example, FVII polypeptides can be administeredto a patient to promote coagulation and reduce blood loss associatedwith surgery and, further, reduce the requirement for blood transfusion.In one embodiment, FVII polypeptides can be administered to subjectsundergoing retropubic prostatectomy. Retropubic prostatectomy is oftenassociated with major blood loss and a subsequent need for transfusion.Subjects undergoing such or similar surgery can be given an intravenousbolus of a therapeutic amount of FVII in the early operative phase toreduce perioperative blood loss by enhancing coagulation at the site ofsurgery. Reduction in blood loss results in elimination of the need forblood transfusion in these patients (Friederich et al. (2003) Lancet361:201-205). FVII polypeptides can be administered to patients withnormal coagulation undergoing other types of surgery to effect rapidhemostasis and prevent blood loss. Non-limiting examples of surgicalprocedures in which FVII, typically administered in the activated form(i.e. FVIIa), can be used a therapy to reduce perioperative bleedinginclude, but are not limited to, cardiac valve surgery (Al Douri et al.(2000) Blood Coag Fibrinol 11:S121-S127), aortic valve replacement(Kastrup et al. (2002) Ann Thorac Surg 74:910-912), resection ofrecurrent hemangiopericytoma (Gerlach et al. (2002) J Neurosurg96:946-948), cancer surgery (Sajdak et al. (2002) Eur J Gynaecol Oncol23:325-326), and surgery on duodenal ulcers (Vlot et al. (2000) Am J Med108:421-423). Treatment with FVII can promote hemostasis at the site ofsurgery and reduce or prevent blood loss, thereby reducing or abolishingthe need for transfusion. The modified FVII polypeptides provided hereinare designed to exhibit enhanced coagulation activity compared withunmodified FVII polypeptides, and might therefore be administered, forexample, at lower doses, less frequently, and with fewer adversereactions. Generally the modified FVII polypeptides are administered asactivated FVII (FVIIa) polypeptides.

Factor VII polypeptides also can be used to promote coagulation andprevent blood loss in subjects with traumatic injury. Trauma is definedas an injury to living tissue by an extrinsic agent, and is the fourthleading cause of death in the United States. Trauma is classified aseither blunt trauma (resulting in internal compression, organ damage andinternal hemorrhage) or penetrative trauma (a consequence of an agentpenetrating the body and destroying tissue, vessel and organs, resultingin external hemorrhaging). Trauma can be caused by several eventsincluding, but not limited to, vehicle accidents (causing blunt and/orpenetrative trauma), gun shot wounds (causing penetrative trauma),stabbing wounds (causing penetrative trauma), machinery accidents(causing penetrative and/or blunt trauma), and falls from significantheights (causing penetrative and/or blunt trauma). Uncontrolledhemorrhage as a result of trauma is responsible for most of theassociated mortality. Diffuse coagulopathy is a relatively commoncomplication associated with trauma patients, occurring in as many as25-36% of subjects. Coagulopathy can develop early after injury,resulting from a variety of factors such as dilution and consumption ofcoagulation factors and platelets, fibrinolysis, acidosis, andhypothermia. Conventional management involves replacement therapy bytransfusion with fresh frozen plasma (FFP) platelets, RBC and/orcryoprecipitate, correcting acidosis, and treating hypothermia. Thesesteps often are insufficient to stop the bleeding and prevent death.Treatment by administration of therapeutic amounts of FVII can promotecoagulation and reduce blood loss in trauma patients. For example, apatient with a gun shot injury presenting with massive blood, inaddition to surgical intervention, be administered FVII to controlcoagulopathic bleeding (Kenet et al. (1999) Lancet 354:1879). Coagulanttherapy with FVII can effectively reduce blood loss and hemorrhage inpatients with blunt and penetrating trauma (Rizoli et al. (2006) Crit.Care 10:R178). The modified FVII polypeptides provided herein aredesigned to exhibit enhanced coagulation activity compared withunmodified FVII polypeptides, and might therefore be administered, forexample, at lower doses, less frequently, and with fewer adversereactions. Generally the modified FVII polypeptides are administered asactivated FVII (FVIIa) polypeptides.

I. Combination Therapies

Any of the modified FVII polypeptides described herein can beadministered in combination with, prior to, intermittently with, orsubsequent to, other therapeutic agents or procedures including, but notlimited to, other biologics, small molecule compounds and surgery. Forany disease or condition, including all those exemplified above, forwhich FVII (including FVIIa and rFVIIa) is indicated or has been usedand for which other agents and treatments are available, FVII can beused in combination therewith. Hence, the modified FVII polypeptidesprovided herein similarly can be used. Depending on the disease orcondition to be treated, exemplary combinations include, but are notlimited to, combination with other plasma purified or recombinantcoagulation factors, procoagulants, such as vitamin K, vitamin Kderivative and protein C inhibitors, plasma, platelets, red blood cellsand corticosteroids.

J. Articles of Manufacture and Kits

Pharmaceutical compounds of modified FVII polypeptides or nucleic acidsencoding modified FVII polypeptides, or a derivative or a biologicallyactive portion thereof can be packaged as articles of manufacturecontaining packaging material, a pharmaceutical composition which iseffective for treating a hemostatic disease or disorder, and a labelthat indicates that modified FVII polypeptide or nucleic acid moleculeis to be used for treating hemostatic disease or disorder.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, for example, U.S. Pat.Nos. 5,323,907, 5,052,558 and 5,033,352, each of which is incorporatedherein in its entirety. Examples of pharmaceutical packaging materialsinclude, but are not limited to, blister packs, bottles, tubes,inhalers, pumps, bags, vials, containers, syringes, bottles, and anypackaging material suitable for a selected formulation and intended modeof administration and treatment. A wide array of formulations of thecompounds and compositions provided herein are contemplated as are avariety of treatments for any hemostatic disease or disorder.

Modified FVII polypeptides and nucleic acid molecules also can beprovided as kits. Kits can include a pharmaceutical compositiondescribed herein and an item for administration. For example a modifiedFVII can be supplied with a device for administration, such as asyringe, an inhaler, a dosage cup, a dropper, or an applicator. The kitcan, optionally, include instructions for application including dosages,dosing regimens and instructions for modes of administration. Kits alsocan include a pharmaceutical composition described herein and an itemfor diagnosis. For example, such kits can include an item for measuringthe concentration, amount or activity of FVII or a FVII regulated systemof a subject.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

K. EXAMPLES Example 1 Cloning and Expression of FVII

A. Cloning of FVII

The nucleotides encoding the 466 amino acid human FVII isoform precursorpolypeptide (P08709; set forth in SEQ ID NO:1) were cloned into themammalian expression vector, pCMV Script (Stratagene; SEQ ID NO:99),which contains a cytomegalovirus (CMV) promoter. Briefly, the CBO-125(SEQ ID NO:100) and CBO-126 (SEQ ID NO:101) oligonucleotides were usedas forward and reverse primers, respectively, to amplify the FVIIsequence by PCR using human FVII cDNA (Invitrogen) as the template. TheCBO-125 primer contained a BamHI restriction site (in bold), a Kozaksequence (double underlined), followed by 18 nucleotides with homologyto the 5′ end of the FVII cDNA sequence (underlined), including the ATGstart codon. The CBO-126 primer contained an EcoRI restriction site (inbold), a stop codon (double underlined) and 21 nucleotides with homologyto the 3′ end of the FVII cDNA sequence (underlined).

CBO-125 forward primer 5′ gcatcatgacgtgacggatcc gccaccatggtctcccaggccctc 3′ CBO-126 reverse primer 5′ gatcgtacgatacgtgaattccta gggaaatggggctcgcaggag 3′

Standard PCR reaction and thermocycling conditions were used inconjunction with the KoD HiFi PCR kit (EMD Biosciences), as recommendedby the manufacturer. The PCR product was digested with BamH I and EcoR Irestriction enzymes and ligated into the BamH I and EcoR I restrictionsites of pCMV Script vector using standard molecular techniques. Thevector was then transformed into Escherichia coli. Selected colonieswere grown and bacterial cells harvested for purification of the plasmidusing routine molecular biology techniques.

B. Generation of FVII Variants

FVII variants were generated using the QuikChange II XL Site-DirectedMutagenesis kit (Stratagene) according to the manufacturersinstructions, with specifically designed oligonucleotides that served asprimers that incorporated a particular mutation into newly synthesizedDNA. The QuikChange method involves linear amplification of template DNAby the PfuUltra high-fidelity DNA polymerase. Complementary primers thatinclude the desired mutation were extended during cycling usingpurified, double-stranded supercoiled pCMV Script vector that containedthe cloned FVII cDNA sequence as a template. Extension of the primersresulted in incorporation of the mutation of interest into the newlysynthesized strands, and resulted in a mutated plasmid with staggerednicks. Following amplification, the nucleic acid was treated with Dpn I,which digests the dam-methylated parental strands of the E. coli-derivedpCMV Script vector. This resulted in “selection” of thenewly-synthesized mutated plasmids, which were not methylated. Thevector DNA containing the desired mutation(s) were transformed intoXL10-Gold ultracompetent E. coli cells, where bacterial ligase repairedthe nicks and allowed normal replication to occur.

Table 13 below sets forth the FVII variants that were generated. In someinstances, FVII variants were generated in which a binding sequence forplatelet integrin α_(IIb)β₃ was inserted in various regions of the FVIIpolypeptide. One of three different integrin α_(IIb)β₃ binding sequenceswere inserted: SFGRGDIRNV (SEQ ID NO:110); CSFGRGDIRNVC (SEQ ID NO:111);or GGGSCSFGRGDIRNVC (SEQ ID NO:112). The integrin α_(IIb)β₃ bindingsequences were inserted at the C-terminus of the FVII polypeptide afteramino acid residue P406 by mature FVII numbering, or inserted bydeletion and replacement of FVII amino acid residues S103 to S111, H115to S126 or T127 to P134 by mature FVII numbering. Other FVII variants inwhich a serum albumin binding sequence was inserted also were generated.These FVII variants contained one of seven different serum albuminbinding sequences: QRLMEDICLPRWGCLWEDDF (SEQ ID NO:103), IEDICLPRWGCLWE(SEQ ID NO:104), DICLPRWGCLWED (SEQ ID NO:105), IEDICLPRWGCLW (SEQ IDNO:106), GGGSIEDICLPRWGCLW (SEQ ID NO:107), DICLPRWGCLWED (SEQ IDNO:108), or GGGSDICLPRWGCLWED (SEQ ID NO:109). The serum albumin bindingsequences were inserted at the C-terminus of the FVII polypeptide afteramino acid residue P406 by mature FVII numbering, or inserted bydeletion and replacement of FVII amino acid residues S103 to S111, H115to S126 or T128 to P134 by mature FVII numbering. The “Gla Swap FIX”FVII variants (i.e. a FVII polypeptide in which the endogenous Gladomain has been replaced with the Gla domain from FIX) contains aminoacid residues Y1 to Y45 of SEQ ID NO:83 at the N-terminus. In someexamples, the “Gla Swap FIX” variants contain one or more amino acidsubstitutions in the FIX Gla domain portion. Mutations that are in theFIX Gla domain portion are enclosed in curly brackets and are referencedusing amino acid positions corresponding to the amino acid positions ofa mature wild-type FIX polypeptide, or the wild-type FIX Gla domain setforth in SEQ ID NO:83. For example, {Gla Swap FIX/M19K} denotes that themodified FVII polypeptide contains a heterologous FIX Gla domain inwhich the methionine at position 19 of the FIX Gla domain set forth inSEQ ID NO:83 is replaced with a lysine. In Table 13 below, the aminoacid residues at which the platelet integrin α_(IIb)β₃ or serum albuminbinding sequence is inserted in the FVII polypeptide, and the amino acidsequence of the binding sequence, are both represented. For example,H115S126delinsQRLMEDICLPRWGCLWEDDF indicates that amino acid residuesH115 thru S126 have been deleted and replaced with a serum albuminbinding sequence with the amino acid sequence QRLMEDICLPRWGCLWEDDF (SEQID NO:103).

TABLE 13 Factor VII Variants FVII poly- Variant Variant peptide SEQ(mature FVII numbering) (Chymotrypsin numbering) ID NO Wild-typeWild-type 3 Q286N Q143N 113 Q286E Q143E 114 Q286D Q143D 115 Q286S Q143S116 Q286T Q143T 117 Q286R Q143R 118 Q286K Q143K 119 Q286A Q143A 120Q286V Q143V 121 Q286M Q143M 122 Q286L Q143L 123 Q286Y Q143Y 124 Gla SwapFIX/Q286R Gla Swap FIX/Q143R 131 H257A/Q286R H117A/Q143R 132 S222A/Q286RS82A/Q143R 133 S222A/H257A/Q286R S82A/H117A/Q143R 134 Gla SwapFIX/S222A/Q286R Gla Swap FIX/S82A/Q143R 135 Gla Swap FIX/H257A/Q286R GlaSwap FIX/H117A/Q143R 136 Gla Swap FIX/S222A/H257A/ Gla SwapFIX/S82A/H117A/ 137 Q286R Q143R Q286R/M298Q Q143R/M156Q 138Q286R/M298Q/K341Q K192Q/Q143R/M156Q 139 K199E/Q286R/M298QK60cE/Q143R/M156Q 140 Gla Swap FIX/Q286R/M298Q Gla Swap FIX/Q143R/M156Q141 Q286R/Q366V Q143R/Q217V 142 Q286R/A292N/A294S/Q366VQ143R/A150N/A152S/Q217V 143 A175S/Q286R/Q366V A39S/Q143R/Q217V 144S222A/Q286R/Q366V S82A/Q143R/Q217V 145 H257S/Q286R H117S/Q143R 146H257S/Q286R/Q366V H117S/Q143R/Q217V 147 S222A/H257A/Q286R/Q366VS82A/H117A/Q143R/Q217V 148 Q286R/H373A Q143R/H224A 149S222A/H257A/Q286R/M298Q S82A/H117A/Q143R/M156Q 150 V158D/E296V/M298QV158D/E296V/M298Q 158 Q286R/K341D Q143R/K192D 151 Q286R/Q366DQ143R/Q217D 152 Q286R/Q366N Q143R/Q217N 153 Q286R/M298Q/Q366DQ143R/M156Q/Q217D 154 Q286R/M298Q/Q366N Q143R/M156Q/Q217N 155Q286R/H373F Q143R/H224F 156 Q286R/M298Q/H373F Q143R/M156Q/H224F 157T239S T99S 159 T239N T99N 160 T239Q T99Q 161 T239V T99V 162 T239L T99L163 T239H T99H 164 T239I T99I 165 P321K P170iK 166 P321E P170iE 167P321Y P170iY 168 P321S P170iS 169 Q366D Q217D 170 Q366E Q217E 171 Q366NQ217N 172 Q366T Q217T 173 Q366S Q217S 174 Q366V Q217V 175 Q366I Q217I176 Q366L Q217L 177 Q366M Q217M 178 H373D H224D 179 H373E H224E 180H373S H224S 181 H373F H224F 182 H373A H224A 183 Q366D/H373E Q217D/H224E184 Q366V/H373V Q217V/H224V 185 Q366V/H373L Q217V/H224L 186 Q366V/H373IQ217V/H224I 187 K161S K24S 188 K161A K24A 189 K161V K24V 190 H216S H76S191 H216A H76A 192 H216K H76K 193 H216R H76R 194 S222A S82A 195 S222KS82K 196 S222V S82V 197 S222D S82D 200 S222N S82N 198 S222E S82E 199H257A H117A 201 H257S H117S 202 S222K/H257A S82K/H117A 203 H216A/H257AH76A/H117A 204 H216A/S222A H76A/S82A 205 S52A S[52]A 206 S60A S[60]A 207E394N/P395A/R396S E245N/P246A/R247S 208 R202S R62S 209 A292N/A294SA150N/A152S 210 G318N G170fN 211 A175S A39S 212 K109N K-26N 213A122N/G124S A[122]N/G[124]S 214 A51N A-84N 215 T130N/E132ST[130]N/E[132]S 216 S50A/S62A S[50]A/S[62]A 217A122N/G124S/E394N/P395A/R396S A[122]N/G[124]S/E245N/P246A/ 218 R247SA122N/G124S/E394N/P395A/R396S/ A[122]N/G[124]S/E245N/P246A/ 219 G318NR247S/G170fN S52N/P54S S[52]N/P[54]S 220 S119N/L121S S[119]N/L[121]S 221T128N/P129A T[128]N/P[129]A 222 Q66N/Y68S Q[66]N/Y[68]S 223S52N/P54S/A122N/G124S/E394N/ S[52]N/P[54]S/A[122]N/G[124]S/ 224P395A/R396S E245N/P246A/R247S K109N/A292N/A294S [K109N]/A150N/A152S 225K109N/A175S [K109N]/A39S 226 V158T/L287T/M298K V21T/L144T/M156K 256V158D/L287T/M298K V21D/L144T/M156K 257 S103S111delinsQRLMEDICLPRWS[103]S[111]delinsQRLMEDICLP 227 GCLWEDDF RWGCLWEDDFH115S126delinsQRLMEDICLPRW H[115]S[126]delinsQRLMEDICL 228 GCLWEDDFPRWGCLWEDDF T128P134delinsQRLMEDICLPRW T[128]P[134]delinsQRLMEDICLP 229GCLWEDDF RWGCLWEDDF S103S111delinsIEDICLPRWGCLS[103]S[111]delinsIEDICLPRWG 230 WE CLWE H115S126delinsIEDICLPRWGCLH[115]S[126]delinsIEDICLPRWG 231 WE CLWE T128P134delinsIEDICLPRWGCLT[128]P[134]delinsIEDICLPRWG 232 WE CLWE S103S111delinsDICLPRWGCLWEDS[103]S[111]delinsDICLPRWGC 233 LWED H115S126delinsDICLPRWGCLWEDH[115]S[126]delinsDICLPRWGC 234 LWED T128P134delinsDICLPRWGCLWEDT[128]P[134]delinsDICLPRWGC 235 LWED P406insIEDICLPRWGCLWP257insIEDICLPRWGCLW 236 P406insGGGSIEDICLPRWGCLWP257insGGGSIEDICLPRWGCLW 237 P406insDICLPRWGCLWED P257insDICLPRWGCLWED238 P406insGGGSDICLPRWGCLWED P257insGGGSDICLPRWGCLWED 239S103S111delinsSFGRGDIRNV S[103]S[111]delinsSFGRGDIRNV 240H115S126delinsSFGRGDIRNV H[115]S[126]delinsSFGRGDIRNV 241T127P134delinsSFGRGDIRNV T[128]P[134]delinsSFGRGDIRNV 242P406insCSFGRGDIRNVC P257insCSFGRGDIRNVC 243 P406insGGGSCSFGRGDIRNVCP257insGGGSCSFGRGDIRNVC 244 Gla Swap FIX/S222A Gla Swap FIX/S82A 245 GlaSwap FIX/H257A Gla Swap FIX/H117A 246 Gla Swap FIX/S222A/H257A Gla SwapFIX/S82A/H117A 247 S222A/M298Q S82A/M156Q 248 H257A/M298Q H117A/M156Q249 S222A/H257A/M298Q S82A/H117A/M156Q 250 S222A/A292N/A294S/Q366VS82A/A150N/A152S/Q217V 251 A175S/S222A/Q366V A39S/S82A/Q217V 252S222A/Q366V S82A/Q217V 253 H257S/Q366V H117S/Q217V 254 S222A/H373AS82A/H224A 255 S103S111delinsIEDICLPRWGCL S[103]S[111]delinsIEDICLPRWG258 WE/G237V CLWE/G97V S103S111delinsDICLPRWGCLWES[103]S[111]delinsDICLPRWGC 259 D/G237V LWED/G97VH115S126delinsQRLMEDICLPRW H[115]S[126]delinsQRLMEDICL 260GCLWEDDF/G237V PRWGCLWEDDF/G97V H115S126delinsIEDICLPRWGCLH[115]S[126]delinsIEDICLPRWG 261 WE/G237V CLWE/G97VH115S126delinsDICLPRWGCLWE H[115]S[126]delinsDICLPRWGC 262 D/G237VLWED/G97V T128P134delinsQRLMEDICLPRW T[128]P[134]delinsQRLMEDICLP 263GCLWEDDF/G237V RWGCLWEDDF/G97V T128P134delinsIEDICLPRWGCLT[128]P[134]delinsIEDICLPRWG 264 WE/G237V CLWE/G97VS103S111delinsQRLMEDICLPRW S[103]S[111]delinsQRLMEDICLP 265GCLWEDDF/G237V RWGCLWEDDF/G97V T128P134delinsDICLPRWGCLWET[128]P[134]delinsDICLPRWGC 266 D/G237V LWED/G97VS103S111delinsSFGRGDIRNV/G237V S[103]S[111]delinsSFGRGDIRNV/ 267 G97VH115S126delinsSFGRGDIRNV/G237V H[115]S[126]delinsSFGRGDIRNV/ 268 G97VT128P134delinsSFGRGDIRNV/G237V T[128]P[134]delinsSFGRGDIRNV/ 269 G97VM298Q/H373F M156Q/H224F 270 S119N/L121S/A175S S[119]N/L[121]S/A39S 271T128N/P129A/A175S T[128]N/P[129]A/A39S 272 A122N/G124S/A175SA[122]N/G[124]S/A39S 273 {GlaSwapFIX/E40L}/Q286R/M298Q{GlaSwapFIX/E[40]L}/Q143R/M156Q 274 {GlaSwapFIX/K43I}/Q286R/M298Q{GlaSwapFIX/K[43]I}/Q143R/M156Q 275 {GlaSwapFIX/Q44S}/Q286R/M298Q{GlaSwapFIX/Q[44]S}/Q143R/M156Q 276 {GlaSwapFIX/M19K}/Q286R/M298Q{GlaSwapFIX/M[19]K}/Q143R/M156Q 277 {GlaSwapFIX/M19K/E40L/K43I/Q44S}/{GlaSwapFIX/M[19]K/E[40]L/K 278 Q286R/M298Q [43]I/Q[44]S}/Q143R/M156QT128N/P129A/Q286R T[128]N/P[129]A/Q143R 279 T128N/P129A/Q286R/M298QT[128]N/P[129]A/Q143R/M156Q 280 T128N/P129A/Q286R/H373FT[128]N/P[129]A/Q143R/H224F 281 V158D/Q286R/E296V/M298QV21D/Q143R/E154V/M156Q 282 T128N/P129A/V158D/E296V/M298QT[128]N/P[129]A/V21D/E154V/M156Q 283 T128N/P129A/S222AT[128]N/P[129]A/S82A 284 GlaSwapFIX/T128N/P129A/S222A/GlaSwapFIX/T[128]N/P[129]A/S82A/ 285 Q286R Q143RGlaSwapFIX/T128N/P129A/Q286R/ GlaSwapFIX/T[128]N/P[129]A/Q143R/ 286M298Q M156Q T128N/P129A/S222A/H257A/Q286R/T[128]N/P[129]A/S82A/H117A/Q143R/ 287 M298Q M156QT128N/P129A/Q286R/M298Q/H373F T[128]N/P[129]A/Q143R/M156Q/ 288 H224FS52A/S60A/V158D/E296V/M298Q S[52]A/S[60]A/V21D/E154V/M156Q 289S52A/S60A/Q286R S[52]A/S[60]A/Q143R 290 S52A/S60A/S222AS[52]A/S[60]A/S82A 291 GlaSwapFIX/S52A/S60A/S222A/Q286RGlaSwapFIX/S[52]A/S[60]A/S82A/ 292 Q143R S52A/S60A/Q286R/M298QS[52]A/S[60]A/Q143R/M156Q 293 S52A/S60A/S222A/H257A/Q286R/S[52]A/S[60]A/S82A/H117A/Q143R/ 298 M298Q M156Q S52A/S60A/Q286R/H373FS[52]A/S[60]A/Q143R/H224F 296 S52A/S60A/Q286R/M298Q/H373FS[52]A/S[60]A/Q143R/M156Q/H224F 297 V158D/T239V/E296V/M298QV21D/T99V/E154V/M156Q 298 T239V/Q286R T99V/Q143R 299 S222A/T239VS82A/T99V 300 GlaSwapFIX/S222A/T239V/Q286R GlaSwapFIX/S82A/T99V/Q143R301 T239V/Q286R/M298Q T99V/Q143R/M156Q 302 S222A/T239V/H257A/Q286R/M298QS82A/T99V/H117A/Q143R/M156Q 303 GlaSwapFIX/T239V/Q286R/M298QGlaSwapFIX/T99V/Q143R/M156Q 304 T239V/Q286R/H373F T99V/Q143R/H224F 305T239V/Q286R/M298Q/H373F T99V/Q143R/M156Q/H224F 306V158D/T239I/E296V/M298Q V21D/T99I/E154V/M156Q 307 T239I/Q286R T99I/Q143R308 S222A/T239I S82A/T99I 309 GlaSwapFIX/S222A/T239I/Q286RGlaSwapFIX/S82A/T99I/Q143R 310 T239I/Q286R/M298Q T99I/Q143R/M156Q 311S222A/T239I/H257A/Q286R/M298Q S82A/T99I/H117A/Q143R/M156Q 312GlaSwapFIX/T239I/Q286R/M298Q GlaSwapFIX/T99I/Q143R/M156Q 313T239I/Q286R/H373F T99I/Q143R/H224F 314 T239I/Q286R/M298Q/H373FT99I/Q143R/M156Q/H224F 315 GlaSwapFIX/S222A/Q286R/H373FGlaSwapFIX/S82A/Q143R/H224F 316 GlaSwapFIX/S222A/Q286R/M298QGlaSwapFIX/S82A/Q143R/M156Q 317 GlaSwapFIX/S222A/Q286R/M298Q/GlaSwapFIX/S82A/Q143R/M156Q/ 318 H373F H224F V158D/E296V/M298Q/H373FV21D/E154V/M156Q/H224F 319 V158D/Q286R/E296V/M298Q/H373FV21D/Q143R/E154V/M156Q/H224F 320 H257A/Q286R/M298Q H117A/Q143R/M156Q 321H257S/Q286R/M298Q H117S/Q143R/M156Q 322 GlaSwapFIX/S222A/H257S/Q286RGlaSwapFIX/S82A/H117S/Q143R 323 S222A/H257S/Q286R/M298QS82A/H117S/Q143R/M156Q 324 H257S/Q286R/M298Q/H373FH117S/Q143R/M156Q/H224F 325 S222A/Q286R/M298Q/H373FS82A/Q143R/M156Q/H224F 326 GlaSwapFIX/Q366V GlaSwapFIX/Q217V 327S222A/Q286R/M298Q S82A/Q143R/M156Q 328 T128N/P129A/A175S/Q366VT[128]N/P[129]A/A39S/Q217V 329 A122N/G124S/A175S/Q366VA[122]N/G[124]S/A39S/Q217V 330 T128N/P129A/A175S/S222AT[128]N/P[129]A/A39S/S82A 331 A122N/G124S/A175S/S222AA[122]N/G[124]S/A39S/S82A 332 T128N/P129A/A175S/Q286RT[128]N/P[129]A/A39S/Q143R 333 A122N/G124S/A175S/Q286RA[122]N/G[124]S/A39S/Q143R 334 GlaSwapFIX/T128N/P129A/A175S/GlaSwapFIX/T[128]N/P[129]A/A39S/ 335 S222A/Q286R S82A/Q143RGlaSwapFIX/A122N/G124S/A175S/ GlaSwapFIX/A[122]N/G[124]S/A39S/ 336S222A/Q286R S82A/Q143R T128N/P129A/A175S/Q286R/M298QT[128]N/P[129]A/A39S/Q143R/M156Q 337 A122N/G124S/A175S/Q286R/M298QA[122]N/G[124]S/A39S/Q143R/M156Q 338 T128N/P129A/A175S/S222A/H257A/T[128]N/P[129]A/A39S/S82A/H117A/ 339 Q286R/M298Q Q143R/M156QA122N/G124S/A175S/S222A/H257A/ A[122]N/G[124]S/A39S/S82A/H117A/ 340Q286R/M298Q Q143R/M156Q T128N/P129A/A175S/Q286R/M298Q/T[128]N/P[129]A/A39S/Q143R/M156Q/ 341 H373F H224FA122N/G124S/A175S/Q286R/M298Q/ A[122]N/G[124]S/A39S/Q143R/M156Q/ 342H373F H224F T128N/P129A/M298Q T[128]N/P[129]A/M156Q 354 {Gla Swap FIX/{Gla Swap FIX/K[43]I}/ 355 K43I}/T128N/P129A/Q286R/M298QT[128]N/P[129]A/Q143R/M156Q T128N/P129A/Q286R/M298Q/Q366NT[128]N/P[129]A/Q143R/M156Q/ 356 Q217N {Gla Swap FIX/ {Gla Swap FIX/ 357K43I}/Q286R/M298Q/Q366N K[43]I}/Q143R/M156QQ217N {Gla Swap FIX/K43I}/{Gla Swap FIX/K[43]I}/ 358 T128N/P129A/Q286R/M298Q/Q366NT[128]N/P[129]A/Q143R/M156Q Q217N T128N/P129A/M298Q/H373FT[128]N/P[129]A/M156Q/H224F 359 V158D/Q286R/E296V/M298QV21D/Q143R/E154V/M156Q 360 M298Q/Q366N/H373F M156Q/Q217N/H224F 361T239V/M298Q/H373F T99V/M156Q/H224F 362 T239I/M298Q/H373FT99I/M156Q/H224F 363 T128N/P129A/Q286R/M298Q/Q366NT[128]N/P[129]A/Q143R/M156Q/ 364 H373F Q217N/H224FT239V/Q286R/M298Q/Q366N T99V/Q143R/M156Q/Q217N 365T239I/Q286R/M298Q/Q366N T99I/Q143R/M156Q/Q217N 366T128N/P129A/T239V/Q286R/M298Q T[128]N/P[129]A/T99V/Q143R/M156Q 367T128N/P129A/S222A/T239V/H257A/ T[128]N/P[129]A/S82A/T99V/H117A/ 368Q286R/M298Q Q143R/M156Q T128N/P129A/T239V/Q286R/M298Q/T[128]N/P[129]A/T99V/Q143R/M156Q/ 369 H373F H224FT128N/P129A/T239I/Q286R/M298Q T[128]N/P[129]A/T99I/Q143R/M156Q 370T128N/P129A/T239I/Q286R/M298Q/ T[128]N/P[129]A/T99I/Q143R/M156Q/ 371H373F H224FC. Expression of FVII Polypeptides

For initial expression analysis by ELISA and Western Blot, FVIIpolypeptides were expressed in BHK-21 cells. For biochemical assays,such as those described below, the FVII polypeptides were expressed inFreestyle™ 293-F cells (Invitrogen).

The wild-type Factor VII polypeptide (SEQ ID NO:3) and variant FVIIpolypeptides were initially expressed in the baby hamster kidney cellline BHK-21 (ATCC CRL 1632). BHK-21 cells were cultured in Eagle'sminimal essential medium (EMEM, Invitrogen) with 10% fetal calf serum(FCS) in 100 mm culture dishes at 37° C. and 5% CO₂. After growth toapproximately 90% confluence, the cells were transfected with 24 μg ofFVII plasmid DNA using the Lipofectamine 2000 kit (Invitrogen) asinstructed by the manufacturer. The media was replaced 6 hours aftertransfection with EMEM without serum containing 1 μg/ml vitamin K1(Sigma) and the cells were incubated for a further 72 hours. Expressionof FVII in the cell culture media was assayed by ELISA or Western Blot.

For subsequent analyses using biochemical assays, the wild-type FactorVII polypeptide (SEQ ID NO:3) and variant FVII polypeptides wereexpressed in Freestyle™ 293-F cells (Invitrogen). Cells were cultured inFreestyle™ 293 media (Invitrogen) at 37° C. and 8% CO₂ in Erlenmeyerflasks with vented caps. The cells were transfected using themanufacturer's suggested protocol. Briefly, after growth to 1×10⁶cells/ml, the cells were centrifuged and the media was exchanged. Thecells were then transfected with 240 μg of FVII plasmid DNA for every240 ml of cells using 293fectin (Invitrogen). In addition, 50 μl of a 1mg/ml stock of Vitamin K₁ (Sigma) in ethanol was added for every 240 mlof cells. The cells were grown for 5 days then the culture supernatantwas harvested. Expression of FVII in the cell culture media was assayedby ELISA.

In some examples, wild-type and variant FVII polypeptides were expressedin CHO-Express (CHOX) cells (Excellgene). CHO Express (CHOX) cell weremaintained in DM202 Complete medium (SAFC BioSciences) and used toinoculate production seed cultures. Seed cultures were grown to 5×10⁶viable cells/mL and approximately 60 mL was used to inoculateapproximately 0.6 L DM202 Complete medium (inoculation density is0.4×10⁶ vc/mL) to generate a production culture. This production culturewas grown for 4 days to reach 8-12×10⁶ vc/mL on the day of transfection.A transfection complex was formed using Factor VII plasmid DNA (6 mg)and 23.1 mg of Polyethylenimine (PEI). The transfection complex was thendiluted in 0.5 L of serum-free Opti-MEM transfection medium(Invitrogen), which was added to the 0.6 L production culture. After 5hours of transfection the culture was further diluted with ˜1 L ProCHO5medium (Lonza) supplemented with 8 mM L-glutamine and 4 mg/L Vitamin K1.The 2.2 L shake flask culture was allowed to express for 5-7 days beforeharvesting the crude Factor VII. Culture supernatants were thenharvested by filtration and FVII was purified.

Expression of one of the FVII variants (Q286R/M298Q) was performed in astable cell line. This line was generated at Excellgene (Monthey,Valais, Switzerland) by transfection of CHOX cells. Briefly, cells weregrown in the presence of methotrexate, then plated by limiting dilutionat 1 cell per well in 96-well plates. Clones producing the highestlevels of variant FVII were determined by ELISA. One clone (clone 52)was further subcloned by a second limiting dilution and plating in96-well plates. The colonies were grown at 37° C. in DM204A media (SAFCBioSciences), supplemented with 8 mM L-glutamine, 1 mM cysteine, 1 mg/Lvitamin K1. Twenty-four clones were found to have higher levels ofQ286R/M298Q expression, by ELISA analysis, than the original clone 52.These 24 clones were further expanded in 6-well plates for 6 days ofgrowth, followed by growth in 40 mL shake flasks for four days. Eachgrowth step was done at 37° C. in DM204A media, supplemented as above.After the four days of growth, clones were frozen at 1×10⁷ viablecells/mL. The levels of Q286R/M298Q produced by each clone weredetermined by ELISA. Clone 5F7 was the highest producer, typicallygenerating 25-35 mg/L Q286R/M298Q.

1. ELISA

An immunoassay was used to quantify the amount of human FVII and FVIIain a sample. Polyclonal antibodies to human FVII were used to captureand detect the protease in the solution. The immunoassay can be used todetermine protein concentration of conditioned medium or a purifiedstock or to determine the concentration of FVII in another sample, forexample, a human or mouse plasma sample. The baseline concentration ofFVII in human blood is approximately 50 nM and the enzymatically activeform, FVIIa, is approximately 1 nM.

To determine the amount of human FVII or FVIIa protein in samples asandwich ELISA was performed. Ninety-six well flat bottom Maxisorpimmuno plates (Nunc) were coated with 100 μl/well of 5 ng/μl avidin(NeutrAvidin, Pierce Biotech.). The plates were covered and incubatedwith shaking for 1 hour at room temperature (RT) followed by washingfour times in PBS with 0.01% Tween-20 (PBST). The plates were blockedfor a minimum of 1 hour at RT with shaking by incubation with 1% bovineserum albumin (BSA) (w/v) in PBS added to each well at 200 μl/well. Theblocked plates were then stored at 4° C. until use (up to 2 weeks).

Before use, the plates were washed four times in PBST to remove the BSA,and 100 μl/well of a 1 ng/μl solution of biotinylated anti-Factor VIIantibody (R&D Systems) was added to each well and the plate wasincubated at room temperature for 45 minutes with shaking to allowcomplexation with the coated avidin. Excess unbound antibody was removedby washing the plate with PBST (four times).

Serial two-fold dilutions of a FVII standard (American Diagnostica;diluted in PBST), ranging from 50 ng/μl to 0.8 ng/μl, were added to theplate at 100 μl/well. A well containing PBST without any FVII also wasincluded as a buffer only control. To assay purified samples (before andafter activation, see Example 3) of FVII or FVIIa, the sample was firstdiluted 1:25 in PBST, and then serial 2-fold dilutions were made so that25-fold, 50-fold, 100-fold and 200-fold dilutions were tested. Thediluted samples were added to the wells in duplicate at 100 μl/well. Toassay plasma samples containing FVII or FVIIa, the plasma sample wasdiluted 1:100 and 1:400 in PBST and added to the wells in duplicate at100 μl/well. A plasma sample without FVII or FVIIa also was included todetermine background levels. The plates were then incubated for 30minutes at RT with shaking to allow for any FVII or FVIIa in the sampleto complex with the anti-FVII antibody.

After incubation with sample, the plates were washed 4 times with PBST.A secondary antibody, Equine anti-human FVII or Murine monoclonalanti-human FVII (American Diagnostica), was diluted 1:5000 in PBST andadded to each well at a volume of 100 μl. The plates were incubated for30 minutes at room temperature with shaking to allow the added antibodyto bind to the FVII or FVII complexes on the plate. To remove excesssecondary antibody, the plates were washed with PBST (4 times). Todetect the bound secondary antibody, 100 μl of goat anti-equine HRPconjugate at a 1:5000 dilution in PBST, or 100 μl of goat anti-mouse HRPconjugate at a 1:20,000 dilution in PBST was added to each well. Afterincubation for 30 minutes at room temperature with shaking, the plateswere washed four times with PBST and 100 μl/well of a solutioncontaining a 1:1 mixture of TMB substrate and hydrogen peroxide solution(Pierce Biotech.) was added. The plates were shaken for approximately 1minute at room temperature before addition of 100 μl/well of 2M H₂SO₄ tostop the reaction. The optical density at 450 nm was measured using aMolecular Device MS Plate reader and the background value for the plate(measured with PBST alone) was subtracted from the measured value fromeach well. A standard curve was generated by plotting the concentrationof the FVII standards versus the absorbance. A standard curve range ofabout 0.2-50 ng/ml was typically generated under the above ELISAconditions. The concentration of each sample was then determined usingthe standard curve and multiplying by the dilution factor, and anaverage and standard deviation was reported.

2. Western Blot

Expression of FVII in cell culture media also was assayed by Westernblot. Aliquots containing the undiluted sample, or two serial 2-folddilutions in PBS, of the cell culture medium from FVII-transfected cells(BHK-21 or CHOX cells) were labeled Conc. 1 (undiluted), Conc. 2 (2-folddilution) and Conc. 3 (4-fold dilution). The samples were loaded on anSDS page gel next to 10, 25, and 50 nanograms of control plasma purifiedrFVII (American Diagnostica). FVII protein produced by BHK-21 or CHOXcells was detected by Western blot using a primary polyclonal equineanti-FVII antibody (American Diagnostica; used at the manufacture'ssuggested concentration) and an HRP-conjugated anti-equine IgG secondaryantibody (a 1:2000 dilution of 1 mg/ml solution from ZymedLaboratories). In some examples, the FVII was detected by Western blotusing a primary rabbit anti-human Factor VIIa antibody (HematologicTechnologies) and an HRP-conjugated anti-rabbit IgG secondary antibody(Invitrogen). Comparison of expression levels was made with the controlplasma purified rFVII. The results show that concentrations ranging fromabout 20 ng to more than 50 ng of FVII was present in the cell culturealiquots.

Example 2 Purification and Activation of FVII Polypeptides

FVII polypeptides were purified using a Q Sepharose Fast Flow, or CaptoQcolumn (GE Healthcare), to which FVII polypeptides with functional Gladomains will adsorb, followed by a calcium elution step. Typically,culture supernatant from the transfected was diluted 2-fold with asolution containing 20 mM Tris pH 8.0 and 0.01% Tween 20, and then 500mM EDTA pH 8.0 was added to the diluted sample to a final concentrationof 1.5 mM. The samples were filtered before being loaded onto the QSepharose Fast Flow or CaptoQ column, which had been pre-equilibratedfirst with Buffer B (20 mM Tris pH 8.0, 1 M NaCl, 0.01% Tween 20), thenBuffer A (20 mM Tris pH 8.0, 0.15 M NaCl, 0.01% Tween 20). After beingloaded, the column was washed with Buffer A until the absorbance of theflow-through at 280 nm reached a baseline. Buffer A was replaced withBuffer C (20 mM Tris pH 8.0, 0.15 M NaCl, 0.01% Tween 20, 5 mM CaCl₂)and a pump wash was performed to completely replace the buffer in thelines. Upon completion of the pump wash, Buffer C was applied to thecolumn at 8 ml/min to elute the FVII polypeptides, which were collectedin fractions. Following elution, the column was washed with Buffer Bwhile still collecting fractions, until the pink pigment (from theculture media) was washed off the column. The column was then washedwith Buffer A to requilibrate it for re-use.

The eluted fractions were further purified using a Mono Q or QHiTrapcolumn (GE Healthcare), which was pre-equilibrated initially with BufferB, and then with Buffer A. The fractions collected with buffer C above,which contained FVII, were pooled and diluted 2-fold with Buffer A,before EDTA, pH 8.0 was added to a final concentration of 40 mM. Smallaliquots (e.g. 100 μl) were optionally taken at this point for analysis,such as by ELISA. The combined sample was loaded onto the Mono Q (orQHiTrap) column, then washed with Buffer A. To elute the bound FVIIpolypeptides, a gradient from 0% to 30% of Buffer B was run through thecolumn and fractions were collected. The column was then washed withBuffer B followed by Buffer A to requilibrate for re-use.

In some examples, after the first Capto Q column, pooled fractions werebuffer exchanged by diafiltration to Buffer D (20 mM MES, pH 6.0, 10 mMCaCl₂, 0.1 M NaCl, 0.01% Tween 20) then loaded onto an SP-HP columnwhich had been pre-equilibrated with Buffer D. After washing with BufferD, a gradient of 0.1 M NaCl to 1.0M NaCl was applied to the column andfractions were collected. Fractions containing FVII were then adjustedto pH 8.0 and diluted 2 fold in Buffer E (20 mM Tris, pH 8.0, 10 mMCaCl₂, 0.01% Tween 20) and applied to a Q H-HP column which had beenpre-equilibrated with Buffer E. This column was then washed with BufferE and the FVII was eluted by a gradient of 0-1 M NaCl in Buffer E.

Purified FVII polypeptides were activated to FVIIa using biotinylatedFactor Xa from the Restriction Protease Factor Xa Cleavage and RemovalKit (Roche). Typically, 7 fractions from the Mono Q purification werepooled in a 15 ml conical tube and 388 μl of 500 mM CaCl₂, 38.9 μl of10% BSA in distilled water, and 3.2 μg of biotinylated Factor Xa wereadded. After incubation for 14-16 hrs at 37° C., 250 μl of ImmobilizedAvidin (Pierce) was added and the sample was mixed at 4° C. for 30minutes. The resulting solution was then filtered through an Econo-pakcolumn (Bio-Rad), and the filtrate was mixed with another 250 μl ofImmobilized Avidin for a further 30 minutes. The solution was filteredagain and the filtrate was concentrated to approximately 300-500 μlusing an Amicon Ultra-4 10 kDa centrifugal filter (Millipore). The FVIIaconcentration was then analyzed by ELISA (as described in Example 1.C.1)and the level of Factor VII activation was monitored by Western blot.Western blotting was performed essentially as described in Example1.C.2, but instead using rabbit anti-human Factor VIIa antibody(Haematologic Technologies, Inc.) at 1:2000 for 1 hr as the primaryantibody, followed by HRP-Goat Anti-Rabbit IgG (H+L) (Invitrogen) at1:5000 for 30 minutes.

Example 3 Determination of the Concentration of Catalytically ViableProtease in a Solution

The concentration of catalytically viable FVIIa in a stock solution wasdetermined by titrating a complex of Factor VIIa and soluble TissueFactor (sTF) with an irreversible peptide inhibitor of FVIIa,Phe-Phe-Arg-Chloromethylketone (FFR-CMK). The inhibitor binds to FVIIabut not to FVII. Extended incubation at a high concentration of FVIIa(50 nM) ensures complete titration of the protease. The residualactivity of the FVIIa/TF complex after incubation with FFR-CMK wasmeasured to determine the concentration of catalytically viable FVIIa inthe original stock solution.

A 96 well clear half area assay plate (Nunc) was pretreated by adding150 μl/well of 1× plate buffer (100 mM Tris pH 8.4, 100 mM NaCl, 0.01%BSA, 0.01% Tween-20) to each well and incubating the plate at 37° C. fora minimum of 1 hour. The buffer was removed completely by blotting on apaper towel and centrifuging the plate upside down to remove anyremaining buffer, and the plate was air-dried for 1 hour and storedcovered at room temperature (RT).

To prepare the FVIIa/sTF/FFR-CMK reaction mixture, a stock of FVIIa(American Diagnostica; diluted to 5 μM in 50% glycerol (v/v) and storedcold in aliquots at −20° C.) or a FVIIa variant was first diluted to 500nM in 1× direct assay buffer (100 mM Tris pH 8.4, 100 mM NaCl, 5 mMCaCl₂, 0.01% BSA). The FVIIa/sTF mixture was then made by mixing 90 μldistilled water with 36 μl 5× direct assay buffer, 18 μl 500 nM FVIIa,and 18 μl 5 μm sTF (recombinant human Coagulation Factor III/solubletissue factor; R&D Systems; the stock solution used was 19.6 μM in 50%glycerol and was diluted to 5 μM in 1× direct assay buffer and stored upto two weeks at 4° C.). The components were then allowed to complex for5 minutes at room temperature.

A stock solution of 10 mM FFR-CMK (BaChem) in DMSO (stored at −20° C.)was diluted in water to 3.5 μM. Using one row of a polypropylene opaquestorage plate (Costar), serial two fold dilutions in water of theFFR-CMK were made across 11 wells of a 96-well opaque plate, with thelast well of the row containing only water as a control. This is the10×FFR-CMK inhibitor series solution. Into each well of a row of thepre-treated 96 well clear half area assay plate, 10.8 μl of theFVIIa/sTF mixture was added, followed by 1.2 μl of the 10×FFR-CMKinhibitor series. The solutions were mixed well and the plate wascentrifuged at <3000 rpm for 5 minutes to remove drips in the wells. Theplate was covered and incubated for 8 hours at 37° C.

To assay the residual activity of the FVIIa/TF complex, a mixture of thesubstrate Spectrozyme FVIIa (American Diagnostica, #217L; reconstitutedstock of 50 μmole vial in 5 mL distilled water to 10 mM and stored at 4°C.) and 5× direct buffer (500 mM Tris pH 8.4, 500 mM NaCl, 25 mM CaCl₂and 0.05% BSA) was first prepared by mixing 360 μl 5× direct assaybuffer with 180 μl of a 10 mM solution of Spectrozyme FVIIa and 1080 μlof water. To each well of the assay plate, 108 μl of the preparedsubstrate solution was added. The wells were mixed and the plate wasincubated at 37° C. The increase in absorbance at 405 nm was measuredevery 30 seconds for one hour at 37° C. on a Spectramax Gemini M5 platereader from Molecular Devices.

Using SoftMax Pro software (Molecular Devices), the absorbance rateswere measured and the fractional activity of proteases incubated with aninhibitor was determined by dividing the measured rate by the rate ofthe uninhibited protease. The fractional activity was graphed againstthe concentration of FFR-CMK, and points that were >90% or <10% of theuninhibited activity were discarded. A line was then drawn through theremaining points to determine the x-intercept, which represents theconcentration of active protease in the solution. The values frommultiple assays was measured and averaged and the standard deviation wasdetermined.

Example 4 Determination of the Catalytic Activity of FVIIa for itsSubstrate, Factor X

The catalytic activity of the FVIIa variants for its substrate, Factor X(FX), was assessed indirectly in a fluorogenic assay by assaying for theactivity of FXa, generated upon activation by FVIIa, on the syntheticsubstrate Spectrafluor FXa.

A. TF-Dependent Catalytic Activity of Wild-Type FVIIa for its Substrate,Factor X

TF-dependent catalytic activity of wild-type FVIIa was assessed in afluorogenic assay in which a lipidated form of purified tissue factor(TF) was included to provide for optimal activity of FVIIa. Enzymeactivity of FXa for Spectrafluor FXa (CH₃SO₂-D-CHA-Gly-Arg-AMC.AcOH) wasdetermined by measuring the increase in absorbance of the generated freefluorophore, AMC (7-amino-4-methylcoumarin), as a function of time.

Briefly, the wild-type FVIIa polypeptide was initially diluted to 0.5 μMin 1× assay buffer (100 mM Tris pH 8.4, 100 mM NaCl, 5 mM CaCl₂, and0.01% BSA), then further diluted to 0.1 nM in assay buffer. Lipidatedfull-length TF (Innovin; Dade Behring) was reconstituted in 20 mL waterto make a 3 nM solution and diluted to 0.2 nM in 1× assay buffer. Fourhundred μl of 0.1 nM FVIIa was mixed with 400 μl 0.2 nM TF and incubatedat room temperature for 5 minutes. The solution was diluted further bytwo, 2-fold dilutions into 1× assay buffer containing 0.2 nM TF toobtain a total of three FVIIa dilutions of 0.05 nM, 0.025 nM, or 0.0125nM FVIIa each containing 0.2 nM TF (FVIIa/TF solutions).

The substrate, Factor X (FX; American Diagnostica; 80 μg) wasreconstituted in 135.6 μl distilled water to give a 10 μM stock andstored in aliquots at −80° C. The aliquots were not frozen and thawedmore than once. The FX stock was diluted to 800 nM in direct assaybuffer, then serially diluted 2-fold to obtain FX solutions ranging from800 nM to 50 nM.

Spectrofluor Xa (American Diagnostica; 10 μmoles) was reconstituted indistilled water to 5 mM and stored at 4° C. To a 96-well black half areaassay plate (Costar), 5 μl Spectrofluor Xa (American Diagnostica) wasadded to each well. Then, 25 μl of the FX solution was added to eachwell. To the last row of wells of the plate, a negative control in whichno FX was added also was included in the assay. In duplicate, the threeconcentrations of the TF/FVIIa solutions were added at 20 μl to wells ofrespective columns of the plate so that each TF/FVIIa dilution wasassayed against each FX dilution, with one set of columns containing noadded TF/FVIIa (i.e. FX alone). The plates were mixed by shaking. Thefluorescence was measured over time with a spectrafluorometer set toread every 30 seconds for 1 hour at 37° C. (Ex: 380 nm, EM: 450 nm,Cut-off: 435 nm), and the time was reported in time squared units.Following the assay, a standard curve of AMC fluorescence in the sameplate reader was generated to covert from fluorescence units to uMsubstrate released in the assay. A 1 mM AMC in DMSO (Invitrogen) wasdiluted to 0.02 mM in 1× assay buffer. Six, two-fold serial dilutions ofthe AMC were made ranging from 20 nM to 0.625 nM in 1× assay buffer. Thefluorescence of the AMC was measured using the same assay conditions asdescribed above and a graph of fluorescence versus concentration of AMCwas plotted. The slope of the line was calculated, which served as theconversion factor for RFU to μM in subsequent calculations.

The kinetics constants for FVIIa activation of FX were calculated byperforming linear regression analysis on the inverse of the substrateconcentration versus the inverse of the velocity of substrate cleavage(in units of seconds 2), with V_(max, FVIIa) calculated as the inverseof the γ-intercept, K_(m, FVIIa) as the slope at the γ-intercept, andV_(max)/K_(m, FVIIa) as the inverse of the slope. The k_(cat) value wasthen derived using the equation;k _(cat) /K _(m, FVIIa) =V _(max) /K _(m, FVIIa)×1/(0.5×k ₂ ×[FVIIa inμM]×(RFU/μM conversion factor))where; k₂=([S]×k_(cat, FXa))/(K_(m, FXa)+[S]), where k_(cat, FXa) andK_(m, FXa) are the constants for FXa cleavage of Spectrofluor Xadetermined experimentally using FXa standards as k_(cat, FXa)=117 sec⁻¹,and K_(m, FXa)=164 μM.

Using the above assay conditions, the kinetic constant k₂ was determinedto be 88.1 sec⁻¹.

The K_(m) and k_(cat) for each of the FVIIa variants was determined toassess the catalytic activity, k_(cat)/K_(m) (M⁻¹sec⁻¹) of each for itssubstrate, FX (Table 14). The wild-type FVIIa protease was assessed andwas found to exhibit an activity of 1.8×10⁷ M⁻¹sec⁻¹. Factor VIIaactivation of Factor X, as measured by Krishnaswamy, et al. (J. Biol.Chem. (1998) 273:8 4378-86) is 2.9×10⁷ M⁻¹sec⁻¹.

B. Analysis of the Catalytic Activity of FVIIa Variants for theSubstrate, Factor X

The catalytic activity of the FVIIa variants for the substrate, Factor X(FX), was assessed indirectly in two types of chromogenic assays byassaying for the activity of FXa, generated upon activation by FVIIa, onthe synthetic substrate Spectrafluor FXa. The two assays were performedeither in the presence or the absence of lipidated tissue factor, toassess both TF-dependent and TF-independent activity. The FVII variantswere expressed, purified and activated to FVIIa as described above inExamples 1 and 2. Although most FVII variants were expressed only inFreestyle™ 293-F cells, some also were expressed in BHK-21 cells.

Lipidated Tissue Factor-Dependent Indirect Assay

The catalytic activity of the FVIIa variants in the presence of tissuefactor was assessed using the assay described in section A of Example 4,above, with minor modifications. One such modification was the use of aFactor X substrate protease that had been treated with ERG-CMK andFFR-CMK to reduce the background activity (Molecular Innovations). Twotypes of data analysis were performed using two separate assays; alinear range analysis assay and a hyperbolic range analysis assay. Thelinear range analysis assay used a range of Factor X concentrationsbetween 0 and 150 nM to ensure accurate measurement of the kineticconstants in the linear range of the dose curve. In contrast, thehyperbolic range analysis assay used a range of Factor X concentrationsbetween 0 and 1.44 μM to ensure accurate measurement of the kineticconstants with a saturating (hyperbolic) dose curve.

The lipidated tissue factor indirect assay with linear range dataanalysis was performed essentially as described in section A of Example4, above, with the following modifications. The FVIIa variant/TFsolutions were prepared as 0.1 nM FVIIa/0.4 nM TF solutions andincubated for 30 minutes before being diluted two-fold in 0.4 nM TF downto a solution containing 1.5625 μM FVIIa/0.4 nM TF. Twenty-five μL ofthe FVIIa/TF solution was mixed with 25 μL of a substrate solution thatcontained 1.0 mM Spectrofluor FXa (American Diagnostica) and one of 300nM, 200 nM, 133.3 nM, 88.9 nM, 59.3, 39.5 nM, 36.3 nM or 0 nM of FactorX (Molecular Innovations). Thus, the final concentrations for the assaywere 0.8 pM FVIIa, 0.2 nM TF, 0.5 mM Spectrofluor FXa and 150 nM, 100nM, 66.7 nM, 44.4 nM, 29.6 nM, 19.8 nM, 13.2 nM or 0 nM of Factor X(Molecular Innovations) in 50 μL/well. The AMC standard curve, whichserved as the conversion factor for RFU to μM in subsequentcalculations, was expanded to include a dose range that covered from 0μM to 100 μM AMC.

The lipidated tissue factor indirect assay with hyperbolic range dataanalysis was performed essentially as described in section A of Example4, above, with the following modifications. The FVIIa variant/TFsolutions were prepared as 0.1 nM FVIIa/0.4 nM TF solutions andincubated for 30 minutes before being diluted two-fold in 0.4 nM TF downto 1.5625 pM (or 0.78 pM for proteases expected to have high activity)FVIIa/0.4 nM TF. Twenty-five μL of the FVIIa/TF solution was mixed with25 μL of a substrate solution that contained 1.0 mM Spectrofluor FXa(American Diagnostica) and one of 1440 nM, 720 nM, 360 nM, 180 nM, 90nM, 45 nM, 22.5 nM or 0 nM of Factor X (Molecular Innovations). Thus,the final concentrations for the assay were 0.8 (or 0.39) pM FVIIa, 0.2nM TF, 0.5 mM Spectrofluor FXa and 7 nM, 720 nM, 360 nM, 180 nM, 90 nM,45 nM, 22.5 nM, 11.25 nM or 0 nM of Factor X (Molecular Innovations) in50 μL/well. The k_(cat) and K_(m) parameters are calculated using theMichaelis Menton hyperbolic equation of the form(V_(max)/(1+(K_(m)/x))). The AMC standard curve, which served as theconversion factor for RFU to μM in subsequent calculations, was expandedto include a dose range that covered from 0 μM to 100 μM AMC.

To determine the kinetic rate constants for the FVIIa or FVIIa variantactivation of FX, raw data collected with the SoftMax Pro application(Molecular Devices) were exported as .XML files. Further data linear andnon-linear analyses were performed with XLfit4, a software package forautomated curve fitting and statistical analysis within the MicrosoftExcel spreadsheet environment (IDBS Software).

For data collected using the linear range assay, the k_(cat)/K_(m)(M⁻¹sec⁻¹) kinetic constants are calculated directly from the slope oflinear regression analyses of the FX concentration versus the velocityof the fluorogenic substrate cleavage (in μM/sec²) wherek_(cat)/K_(m)=slope/[FVIIa]×0.5×k₂. The correction factor k₂ wasdetermined to be 45 using the method described in section A of Example 4and kinetic constants for FXa cleavage of Spectrofluor FXa ofk_(cat,FXa)=56 sec⁻¹ and K_(m, FXa)=126 nM, determined experimentallywith activated FX (FXa) that was previously active site titrated withAT-III/heparin. Excluding data points that resulted in R² values lessthan 0.98 ensured the linearity of the data sets used in the fittingroutine.

Analyses of data collected using the hyperbolic range assay werecalculated from non-linear regression analyses of the FX concentrationversus the velocity of the fluorogenic substrate cleavage (in μM/sec²).The individual k_(cat) and K_(m) parameters are calculated as fitparameters using the Michaelis Menton hyperbolic equation of the form(V_(max)/(1+(K_(m)/x))) where k_(cat)=V_(max)/[FVIIa]×0.5×k₂. Thekinetic constant, k_(cat)/K_(M) was calculated from the individualk_(cat) and K_(m) fitted parameters.

Tissue Factor-Independent Indirect Assay

The catalytic activity of the FVIIa variants in the presence of tissuefactor was assessed in an indirect assay similar to that described aboveexcept that tissue factor was not included in the assay. Thus, the assayto assess TF-independent activity was performed essentially as describedabove, with the following modifications. The FVIIa variant solutionswere diluted to 50 nM (or 5 nM for variants expected to have highTF-independent activity). Twenty-five μL of each FVIIa solution wasmixed with 25 μL of a substrate solution that contained 1.0 mMSpectrofluor FXa (American Diagnostica) and one of 1050 nM, 700 nM,466.7 nM, 311.1 nM, 207.4 nM, 138.3 nM, 92.2 nM or 0 nM of Factor X(Molecular Innovations). Thus, the final concentrations for the assaywere 25 nM FVIIa (or 2.5 nM for high activity variants), 0.5 mMSpectrofluor FXa and 525 nM, 350 nM, 233.3 nM, 155.6 nM, 103.7 nM, 69.1nM, 46.1 nM or 0 nM of Factor X (Molecular Innovations) in 50 μL/well.Data analyses were performed as described for the linear range assay,above with no modifications.

Table 14 provides the catalytic activity of FVIIa variants as measuredin a TF-dependent Indirect Assay using FVIIa polypeptides expressed from293-F cells and BHK-21 cells, and the catalytic activity as measured ina TF-independent Indirect Assay using FVIIa polypeptides expressed from293-F cells and/or BHK-21 cells. The results are presented as thekinetic constant for catalytic activity, k_(cat)/K_(m) (M⁻¹sec⁻¹), andalso expressed as a percentage of the activity of the wild-type FVIIa,wherein the activity is catalytic activity, k_(at)/K_(m) (M⁻¹sec⁻¹) ofeach FVIIa variant for its substrate, FX. The use of the linear orhyperbolic range data analysis also is indicated for the valuespresented in the tables. Not all FVIIa variants were assayed in eachassay. Several FVIIa variants exhibited increased catalytic activitycompared to the wild-type FVIIa molecule. For example, the FVIIapolypeptide containing just the Q286R mutation (Q286R-FVIIa), has acatalytic activity of between 2 and 3 times that of wild-type FVIIa, andthe FVIIa polypeptide containing the Q286R and M298Q mutations(Q286R/M298Q-FVIIa), has a catalytic activity of over 3 times that ofwild-type FVIIa.

TABLE 14 Catalytic activity of FVIIa variants Mutation Mutationk_(cat)/K_(M) k_(cat)/K_(M) (mature FVII numbering) (Chymotrypsinnumbering) Assay Format (M⁻¹s⁻¹) (% WT) TF-Dependent Indirect Assay withFVIIa polypeptides from 293-F cells Q286N Q143N hyperbolic 4.88 × 10⁷100 Q286E Q143E hyperbolic 1.14 × 10⁷ 23 Q286D Q143D hyperbolic 6.04 ×10⁶ 12 Q286S Q143S hyperbolic 4.64 × 10⁷ 95 Q286T Q143T hyperbolic 2.44× 10⁷ 50 Q286R Q143R linear 1.11 × 10⁸ 323 Q286K Q143K hyperbolic 5.44 ×10⁷ 112 Q286A Q143A hyperbolic 8.55 × 10⁷ 175 Q286V Q143V hyperbolic1.65 × 10⁷ 34 H216S H76S linear 4.74 × 10⁷ 138 H216A H76A linear 5.98 ×10⁷ 175 H216K H76K hyperbolic 6.51 × 10⁷ 133 H216R H76R hyperbolic 9.44× 10⁷ 193 S222A S82A linear 5.73 × 10⁷ 167 S222K S82K linear 8.02 × 10⁷234 H257A H117A linear 3.90 × 10⁷ 114 H257S H117S linear 5.90 × 10⁷ 172K161S K24S hyperbolic 5.99 × 10⁷ 123 K161A K24A linear 4.22 × 10⁷ 123K161V K24V hyperbolic 5.45 × 10⁷ 112 H373D H224D linear 1.79 × 10⁷ 52H373E H224E linear 2.79 × 10⁷ 81 H373S H224S linear 2.75 × 10⁷ 80 H373FH224F linear 5.11 × 10⁷ 149 H373A H224A linear 3.11 × 10⁷ 91 S52A S[52]Alinear 4.66 × 10⁷ 136 S60A S[60]A linear 5.15 × 10⁷ 150 Q366D Q217Dlinear 1.88 × 10⁷ 55 Q366E Q217E linear 4.77 × 10⁷ 139 Q366N Q217Nlinear 5.64 × 10⁷ 165 Q366T Q217T linear 3.42 × 10⁷ 100 Q366S Q217Slinear 2.70 × 10⁷ 79 Q366V Q217V linear 6.59 × 10⁷ 192 E394N/P395A/R396SE245N/P246A/R247S linear 5.32 × 10⁷ 155 R202S R62S linear 2.57 × 10⁷ 75A292N/A294S A150N/A152S linear 0 0 G318N G170fN linear 5.50 × 10⁷ 161A175S A39S linear 3.32 × 10⁷ 97 K109N K[109]N linear 5.97 × 10⁷ 174A122N/G124S A[122]N/G[124]S linear 5.27 × 10⁷ 154 T130N/E132ST[130]N/E[132]S linear 6.35 × 10⁷ 185 A122N/G124S/A[122]N/G[124]S/E245N/P246A/ linear 4.88 × 10⁷ 142 E394N/P395A/R396SR247S V158T/L287T/M298K V21T/L144T/M156K linear 4.50 × 10⁶ 13V158D/L287T/M298K V21D/L144T/M156K linear 4.48 × 10⁶ 13S103S111delinsSFGRGDIRNV S[103]S[111]delinsSFGRGDIR linear 4.83 × 10⁷141 NV P406insCSFGRGDIRNVC P257insCSFGRGDIRNVC linear 6.16 × 10⁷ 180P406insGGGSCSFGRGDIRNVC P257insGGGSCSFGRGDIRN linear 7.47 × 10⁷ 218 VCT128N/P129A T[128]N/P[129]A linear 5.96 × 10⁷ 174 S222A/Gla Swap FIXS82A/Gla swap FIX linear 6.55 × 10⁷ 189 H257A/Gla Swap FIX H117A/Glaswap FIX linear 6.45 × 10⁷ 186 S222A/H257A/Gla Swap FIX S82A/H117A/Glaswap FIX linear 5.77 × 10⁷ 168 Q286R/Gla Swap FIX Q143R/Gla swap FIXlinear 1.11 × 10⁸ 323 Q286R/H257A Q143R/H117A linear 1.27 × 10⁸ 371Q286R/S222A Q143R/S82A linear 1.42 × 10⁸ 415 Q286R/S222A/H257AQ143R/S82A/H117A linear 9.51 × 10⁷ 278 Q286R/S222A/Gla Swap FIXQ143R/S82A/Gla swap FIX linear 1.61 × 10⁸ 470 Q286R/H257A/Gla Swap FIXQ143R/H117A/Gla swap FIX linear 8.09 × 10⁷ 234 Q286R/S222A/H257A/GlaSwap Q143R/S82A/H117A/Gla swap linear 7.75 × 10⁷ 226 FIX FIXQ286R/M298Q/K341Q Q143R/M156Q/K192Q linear 3.93 × 10⁷ 115Q286R/M298Q/K199E Q143R/M156Q/K60cE linear 7.74 × 10⁷ 226 T239S T99Slinear 1.74 × 10⁷ 51 T239Q T99Q linear 1.74 × 10⁷ 51 T239V T99V linear9.57 × 10⁷ 279 T239L T99L linear 3.77 × 10⁷ 110 T239H T99H linear 9.90 ×10⁶ 29 T239I T99I linear 3.50 × 10⁷ 102 S222A/H257A/M298QS82A/H117A/M156Q linear 7.75 × 10⁷ 224 S222A/H257A/Q286R/M298QS82A/H117A/Q143R/M156Q linear 2.00 × 10⁸ 583 S222A/H257A S82A/H117Alinear 5.02 × 10⁷ 147 A175S/Q286R/Q366V A39S/Q143R/Q217V linear 8.08 ×10⁷ 236 A175S/S222A/Q366V A39S/S82A/Q217V linear 3.78 × 10⁷ 109K109N/A175S K[109]N/A39S linear 3.67 × 10⁷ 107 S222A/Q286R/Q366VS82A/Q143R/Q217V linear 1.27 × 10⁸ 369 Q286M Q143M linear 5.25 × 10⁷ 153Q286L Q143L linear 2.02 × 10⁷ 59 Q286Y Q143Y linear 1.61 × 10⁷ 47 Q366IQ217I linear 9.37 × 10⁷ 274 Q366L Q217L linear 6.87 × 10⁷ 201 Q366MQ217M linear 6.61 × 10⁷ 193 S222V S82V linear 6.04 × 10⁷ 176 S222D S82Dlinear 5.34 × 10⁷ 156 S222N S82N linear 6.82 × 10⁷ 199 S222E S82E linear5.48 × 10⁷ 160 H216A/H257A H76A/H117A linear 6.62 × 10⁷ 193 H216A/S222AH76A/S82A linear 5.46 × 10⁷ 159 H257S/Q286R H117S/Q143R linear 3.93 ×10⁷ 115 H257S/Q366V H117S/Q217V linear 6.71 × 10⁷ 194 H257S/Q286R/Q366VH117S/Q143R/Q217V linear 1.58 × 10⁸ 457 S222A/H257A/Q286R/Q366VS82A/H117A/Q143R/Q217V linear 1.86 × 10⁸ 538 Q366V/H373V Q217V/H224Vlinear 1.84 × 10⁷ 53 Q366V/H373L Q217V/H224L linear 3.07 × 10⁷ 89Q286R/H373A Q143R/H224A linear 5.89 × 10⁷ 172 S222A/H373A S82A/H224Alinear 3.64 × 10⁷ 106 Q286R/M298Q/K341D Q143R/M156Q/K192D linear 1.18 ×10⁷ 34 Q286R/K341D Q143R/K192D linear 1.11 × 10⁷ 32 Q286R/Q366DQ143R/Q217D linear 1.53 × 10⁷ 45 Q286R/Q366N Q143R/Q217N linear 5.42 ×10⁷ 158 Q286R/M298Q/Q366D Q143R/M156Q/Q217D linear 1.91 × 10⁷ 56Q286R/M298Q/Q366N Q143R/M156Q/Q217N linear 1.04 × 10⁸ 305 Q286R/H373FQ143R/H224F linear 9.08 × 10⁷ 265 Q286R/M298Q/H373F Q143R/M156Q/H224Flinear 1.51 × 10⁸ 440 M298Q/H373F M156Q/H224F linear 8.49 × 10⁷ 248S119N/L121S/A175S S[119]N/L[121]S/A39S linear 2.92 × 10⁷ 85T128N/P129A/A175S T[128]N/P[129]A/A39S linear 2.98 × 10⁷ 87A122N/G124S/A175S A[122]N/G[124]S/A39S linear 2.79 × 10⁷ 81 M298Q M156Qlinear  1.4 × 10⁸ 409 TF-Dependent Indirect Assay with FVIIapolypeptides from BHK-21 cells WT WT linear 5.42 × 10⁷ 100 Q286R Q143Rlinear 1.01 × 10⁸ 187 H216A H76A linear 5.98 × 10⁷ 110 S222A S82A linear6.42 × 10⁷ 118 H257A H117A linear 4.96 × 10⁷ 91 H257S H117S linear 7.65× 10⁷ 141 H373F H224F linear 4.82 × 10⁷ 89 S52A S[52]A linear 3.50 × 10⁷65 S60A S[60]A linear 3.22 × 10⁷ 59 Q366D Q217D linear 9.80 × 10⁶ 18Q366N Q217N linear 3.44 × 10⁷ 63 Q366V Q217V linear 1.86 × 10⁸ 342 G318NG170fN linear 5.46 × 10⁷ 101 A175S A39S linear 2.12 × 10⁷ 39 A122N/G124SA[122]N/G[124]S linear 8.05 × 10⁷ 148 A51N A[51]N linear 1.02 × 10⁸ 188S52A/S60A S[52]A/S[60]A linear 1.05 × 10⁸ 193 P406insGGGSCSFGRGDIRNVCP257insGGGSCSFGRGDIRN linear 9.54 × 10⁷ 176 VC S119N/L121SS[119]N/L[121]S linear 5.75 × 10⁷ 106 T128N/P129A T[128]N/P[129]A linear8.76 × 10⁷ 161 Q286R/S222A Q143R/S82A linear 1.24 × 10⁸ 229Q286R/S222A/H257A Q143R/S82A/H117A linear 1.06 × 10⁸ 196 Q286R/S222A/GlaSwap FIX Q143R/S82A/Gla swap FIX linear 8.52 × 10⁷ 157 Q286R/M298QQ143R/M156Q linear 1.85 × 10⁸ 341 Q286R/M298Q/K341Q Q143R/M156Q/K192Qlinear 3.11 × 10⁷ 57 Q286R/M298Q/K199E Q143R/M156Q/K60cE linear 9.18 ×10⁷ 169 P321K P170iK linear 3.43 × 10⁷ 63 P321E P170iE linear 5.59 × 10⁷103 P321Y P170iY linear 4.48 × 10⁷ 83 P321S P170iS linear 5.53 × 10⁷ 102T239N T99N linear 1.64 × 10⁷ 30 T239Q T99Q linear 1.70 × 10⁷ 31 T239VT99V linear 9.81 × 10⁷ 181 T239L T99L linear 5.24 × 10⁷ 97 T239H T99Hlinear 1.25 × 10⁷ 23 T239I T99I linear 4.67 × 10⁷ 86 S222A/M298QS82A/M156Q linear 7.13 × 10⁷ 131 H257A/M298Q H117A/M156Q linear 1.28 ×10⁸ 236 S222A/H257A/Q286R/M298Q S82A/H117A/Q143R/M156Q linear 1.94 × 10⁸358 Q286R/M298Q/Gla Swap FIX Q143R/M156Q/Gla swap FIX linear 2.64 × 10⁸487 Q286R/Q366V Q143R/Q217V linear 7.92 × 10⁷ 146 A175S/Q286R/Q366VA39S/Q143R/Q217V linear 7.63 × 10⁷ 141 K109N/A175S K[109]N/A39S linear2.45 × 10⁷ 45 S222A/Q286R/Q366V S82A/Q143R/Q217V linear 1.44 × 10⁸ 265Q286R/M298Q/K341D Q143R/M156Q/K192D linear 1.35 × 10⁷ 25 Q286R/H373FQ143R/H224F linear 1.18 × 10⁸ 218 Q286R/M298Q/H373F Q143R/M156Q/H224Flinear 2.01 × 10⁸ 371 M298Q/H373F M156Q/H224F linear 8.69 × 10⁷ 160A122N/G124S/A175S A[122]N/G[124]S/A39S linear 1.93 × 10⁷ 36 M298Q M156Qlinear 9.34 × 10⁷ 172 TF-Independent Indirect Assay Mutation Mutation293-F Cells BHK-21 Cells (mature FVII (Chymotrypsin k_(cat)/K_(M)k_(cat)/K_(M) k_(cat)/K_(M) k_(cat)/K_(M) numbering) numbering) (M⁻¹s⁻¹)(% WT) (M⁻¹s⁻¹) (% WT) WT WT 2.26 × 10¹ 100 1.58 × 10¹ 100 Q286N Q143N3.03 × 10¹ 134 Q286E Q143E 4.80 21 Q286D Q143D   3.50 × 10⁻¹ 2 Q286SQ143S 2.66 × 10¹ 118 Q286T Q143T 1.51 × 10¹ 67 Q286R Q143R 4.87 × 10¹215 4.08 × 10¹ 259 Q286K Q143K 3.95 × 10¹ 175 Q286A Q143A 2.11 × 10¹ 93Q286V Q143V 2.35 10 S222A S82A 7.36 × 10¹ 326 3.10 × 10¹ 197 H257A H117A2.02 × 10¹ 89 1.18 × 10¹ 75 H257S H117S 1.75 × 10¹ 77 1.33 × 10¹ 84Q366D Q217D 6.30 28 2.30 15 Q366E Q217E 2.38 × 10¹ 105 Q366N Q217N 2.26× 10¹ 100 1.36 × 10¹ 86 Q366T Q217T 2.48 × 10¹ 110 Q366S Q217S 1.02 ×10¹ 45 Q366V Q217V 2.90 × 10¹ 128 8.36 × 10¹ 530 A51N A[51]N 2.07 × 10¹91 V158T/L287T/M298K V21T/L144T/M156K 4.65 21 V158D/L287T/M298KV21D/L144T/M156K 2.50 11 S52A/S60A S[52]A/S[60]A 1.68 × 10¹ 106T128N/P129A T[128]N/P[129]A 1.43 × 10¹ 91 Q286R/Gla Swap FIX Q143R/Glaswap FIX 4.37 × 10¹ 193 Q286R/H257A Q143R/H117A 1.07 × 10¹ 47Q286R/S222A Q143R/S82A 1.00 × 10² 444 3.18 × 10¹ 202 Q286R/S222A/H257AQ143R/S82A/H117A 9.60 × 10 61 Q286R/S222A/Gla Swap Q143R/S82A/Gla swap1.82 × 10² 804 3.63 × 10¹ 230 FIX FIX Q286R/S222A/H257A/GlaQ143R/S82A/H117A/Gla 2.79 × 10¹ 123 Swap FIX swap FIX Q286R/M298QQ143R/M156Q 3.02 × 10² 1916 Q286R/M298Q/K341Q Q143R/M156Q/K192Q 1.50 ×10² 665 3.65 × 10² 2319 Q286R/M298Q/K199E Q143R/M156Q/K60cE 8.69 × 10¹385 2.29 × 10² 1451 P321K P170iK 1.13 × 10¹ 71 S222A/M298Q S82A/M156Q7.85 × 10² 4981 H257A/M298Q H117A/M156Q 4.12 × 10¹ 262S222A/H257A/Q286R/M298Q S82A/H117A/Q143R/M156Q 6.09 × 10² 2695 1.90 ×10² 1208 Q286R/M298Q/Gla Swap Q143R/M156Q/Gla swap 7.52 × 10² 4775 FIXFIX Q286R/Q366V Q143R/Q217V 2.38 × 10¹ 151 A175S/Q286R/Q366VA39S/Q143R/Q217V 3.87 × 10¹ 171 1.23 × 10¹ 78 S222A/Q286R/Q366VS82A/Q143R/Q217V 3.21 × 10¹ 204 Q286M Q143M 1.07 × 10¹ 47 Q286L Q143L3.20 14 Q286Y Q143Y   9.50 × 10⁻¹ 4 Q366I Q217I 6.29 × 10¹ 278 Q366LQ217L 2.54 × 10¹ 112 Q366M Q217M 4.05 × 10¹ 179 Q286R/K341D Q143R/K192D1.80 8 Q286R/Q366D Q143R/Q217D 1.00 4 Q286R/Q366N Q143R/Q217N 2.75 12Q286R/M298Q/Q366D Q143R/M156Q/Q217D 6.80 30 Q286R/M298Q/Q366NQ143R/M156Q/Q217N 2.12 × 10¹ 94 Q286R/H373F Q143R/H224F 2.20 × 10¹ 139Q286R/M298Q/H373F Q143R/M156Q/H224F 2.16 × 10² 957 3.17 × 10² 2009M298Q/H373F M156Q/H224F 2.36 × 10² 1499 M298Q M156Q 4.59 × 10² 2029  3.1× 10² 1969

In a further set of experiments, the catalytic activity of FVIIapolypeptides produced in BHK-21 cells was analyzed using theTF-independent and TF-dependent indirect assays described above withminor modifications. Several variants were produced in CHOX cells inaddition to BHK-21 cells or exclusively in CHOX cells. The variants wereassayed under identical conditions regardless of the celline used. Forthe TF-dependent catalytic assay (using linear analysis), the FVIIapolypeptides were first active site titrated with 4-methylumbelliferylp′-guanidinobenzoate (MUGB) to determine the FVIIa concentration, asdescribed in Example 12, below. To maximize the number of data points inthe linear range, the maximal concentration of FX in the assay was setto 25 nM (ie. 0-25 nM instead of 0-150 nM). The FX used in the assay wasactivated (i.e. FXa) and titrated withfluorescein-mono-p′-guanidinobenzoate (FMGB), as described in Example15, below. The kinetic constants for cleavage of Spectrafluor FXasubstrate were determined on this active site titrated FXa anddemonstrated to be: K_(m) of 190.2 μM and a k_(cat) of 340 s⁻¹. Theprimary difference being in k_(cat) and mostly due to the improvedactive site determinations. These parameters give a revised k₂correction factor value of 246.4 that is used in the linear analysis todetermine the catalytic activity of the FVIIa polypeptides in thepresence of TF.

For the TF-independent catalytic assay, the FVIIa polypeptides werefirst active site titrated with 4-methylumbelliferylp′-guanidinobenzoate (MUGB) to determine the FVIIa concentration, asdescribed in Example 12, below. The FX used in the assay was activated(i.e. FXa) and titrated with fluorescein-mono-p′-guanidinobenzoate(FMGB), as described in Example 15. The kinetic constants for cleavageof Spectrafluor FXa substrate were determined on this active sitetitrated FXa and demonstrated to be: K_(m) of 190.2 μM and a k_(cat) of340 s⁻¹. These parameters give a revised k₂ correction factor value of246.4 that is used in the analysis to determine the catalytic activityof the FVIIa polypeptides in the absence of TF.

Table 15 sets forth the catalytic activity of each of the FVIIa variantpolypeptides assayed. The results are presented as the kinetic constantfor catalytic activity, k_(cat)/K_(m) (M⁻¹sec⁻¹), and also expressed asa percentage of the activity of the wild-type FVIIa, wherein theactivity is catalytic activity, k_(cat)/K_(m) (M⁻¹sec⁻¹) of each FVIIavariant for its substrate, FX. The standard deviation (SD), coefficientof variation (as a percentage; % CV) and the number of assays performed(n) also are provided. Some of the variants displayed markedly increasedcatalytic activity compared to the wildtype FVII polypeptide. Forexample, the Gla swap FIX/Q286R/M298Q variant exhibited a TF-dependentcatalytic activity over 6 times that of the wild type FVII polypeptide.The increased catalytic activity of the FVIIa variants was morepronounced in the TF-independent assay. For example, the GlaswapFIX/Q366V variants had over 9 times more catalytic activity thanwild-type FVIIa, the Gla swap FIX/Q286R/M298Q, {Gla swapFIX/E40L}/Q286R/M298Q, {Gla swap FIX/K43I}/Q286R/M298Q, and {Gla swapFIX/Q44S}/Q286R/M298Q variants had over 70-80 times more catalyticactivity than wild-type FVIIa, and the S52A/S60A/V158D/E296V/M1298Qvariant had over 220 times more catalytic activity than wild-type FVIIa.

TABLE 15 Catalytic activity of FVIIa variants Mutation Mutationk_(cat)/K_(M) k_(cat)/K_(M) (mature FVII numbering) (Chymotrypsinnumbering) (M⁻¹s⁻¹) SD % CV (% WT) n TF-dependent assay WT (NovoSeven ®)WT (NovoSeven ®) 3.98E+07 1.02E+07 26% 106% 30 WT (NovoSeven-RT ®) WT(NovoSeven-RT ®) 3.48E+07 8.33E+06 24% 93% 10 WT WT 3.75E+07 5.44E+0615% 100% 13 WT† WT† 3.76E+07 7.09E+06 19% 100% 10 T128N/P129AT[128]N/P[129]A 4.65E+07 1.09E+07 23% 124% 5 Gla swap FIX Gla swap FIX5.38E+07 9.08E+05 2% 144% 2 K109N K[109]N 5.54E+07 8.57E+06 15% 148% 2A122N/G124S A[122]N/G[124]S 3.87E+07 4.96E+06 13% 103% 2 S52A/S60AS[52]A/S[60]A 3.56E+07 4.63E+06 13% 95% 2 M298Q M156Q 6.76E+07 7.38E+0611% 180% 6 M298Q† M156Q† 7.46E+07 9.42E+06 13% 198% 4 T128N/P129A/M298Q†T[128]N/P[129]A/M156Q† 6.29E+07 1.28E+07 20% 167% 4 V158D/E296V/M298QV21D/E154V/M156Q 1.81E+08 4.43E+07 25% 482% 8 V158D/E296V/M298Q†V21D/E154V/M156Q† 1.65E+08 4.08E+07 25% 441% 10 T128N/P129A/V158D/E296V/T[128]N/P[129]A/V21D/E154V/ 2.01E+08 1.54E+07 8% 537% 4 M298Q M156QS52A/S60A/V158D/E296V/ S[52]A/S[60]A/V21D/E154/ 2.00E+08 4.31E+05 0%532% 2 M1298Q M156Q Q286R Q143R 8.06E+07 1.43E+07 18% 215% 5T128N/P129A/Q286R T[128]N/P[129]A/Q143R 8.45E+07 1.90E+07 22% 226% 6T128N/P129A/Q286R† T[128]N/P[129]A/Q143R† 6.20E+07 165% 1S52A/S60A/Q286R S[52]A/S[60]A/Q143R 4.10E+07 6.71E+06 16% 109% 4 S222AS82A 4.07E+07 1.17E+07 29% 109% 4 T128N/P129A/S222A T[128]N/P[129]A/S82A6.25E+07 6.78E+06 11% 167% 4 S52A/S60A/S222A S[52]A/S[60]A/S82A 3.91E+078.75E+06 22% 104% 3 H257S H117S 1.18E+08 2.02E+07 17% 316% 2 H373F H224F5.58E+07 2.05E+07 37% 149% 2 Q366V Q217V 5.48E+07 2.69E+06 5% 146% 2 GlaswapFIX/Q366V Gla swapFIX/Q217V 9.11E+07 2.50E+07 27% 243% 3 A175S A39S2.11E+07 6.10E+06 29% 56% 3 K109N/A175S K[109]N/A39S 1.74E+07 4.29E+0625% 46% 5 S119N/L121S/A175S S[119]N/L[121]S/A39S 1.73E+07 1.28E+06 7%46% 2 T128N/P129A/A175S T[128]N/P[129]A/A39S 8.59E+06 1.82E+06 21% 23% 2A122N/G124S/A175S A[122]N/G[124]S/A39S 1.05E+07 1.12E+06 11% 28% 2Q286R/H257A Q143R/H117A 9.91E+07 1.74E+07 18% 264% 2 Q286R/H257A†Q143R/H117A† 3.08E+07 1.52E+07 49% 82% 4 Q286R/S222A Q143R/S82A 1.11E+083.21E+07 29% 296% 4 Gla swap FIX/ Gla swap FIX/ 1.47E+08 2.53E+07 17%393% 3 T128N/P129A/ T[128]N/P[129]A/ S222A/Q286R S82A/Q143R Gla swapFIX/ Gla swap FIX/ 1.43E+08 1.63E+07 11% 379% 2 T128N/P129A/T[128]N/P[129]A/ S222A/Q286R† S82A/Q143R† Gla swap FIX/ Gla swap FIX/7.24E+07 2.36E+06 3% 193% 2 S52A/S60A/S222A/Q286RS[52]A/S[60]A/S82A/Q143R Q286R/S222A/H257A Q143R/S82A/H117A 6.98E+071.64E+07 23% 186% 3 Q286R/M298Q Q143R/M156Q 1.66E+08 3.86E+07 23% 442%14 Q286R/M298Q† Q143R/M156Q† 1.34E+08 2.37E+07 18% 356% 15 Q286R/M298Q§Q143R/M156Q§ 1.54E+08 3.86E+07 25% 408% 6 Gla swap FIX/ Gla swap FIX/2.55E+08 6.16E+07 24% 680% 6 Q286R/M298Q Q143R/M156Q Gla swap FIX/ Glaswap FIX/ 2.30E+08 5.10E+07 22% 613% 4 Q286R/M298Q† Q143R/M156Q†T128N/P129A/Q286R/ T[128]N/P[129]A/Q143R/ 1.86E+08 2.64E+07 14% 497% 6M298Q M156Q T128N/P129A/Q286R/ T[128]N/P[129]A/Q143R/ 1.50E+08 4.16E+0728% 398% 4 M298Q† M156Q† Gla swap FIX/ Gla swap FIX/ 2.11E+08 4.41E+0721% 562% 3 T128N/P129A/Q286R/ T[128]N/P[129]A/Q143R/ M298Q M156Q Glaswap FIX/ Gla swap FIX/ 1.99E+08 6.79E+07 34% 529% 5 T128N/P129A/Q286R/T[128]N/P[129]A/Q143R/ M298Q† M156Q† {Gla swap FIX/E40L}/ {Gla swapFIX/E[40]L}/ 2.08E+08 4.39E+07 21% 556% 4 Q286R/M298Q Q143R/M156Q {Glaswap FIX/K43I}/ {Gla swap FIX/K[43]I}/ 2.73E+08 5.21E+07 19% 727% 5Q286R/M298Q Q143R/M156Q {Gla swap FIX/K43I}/ {Gla swap FIX/K[43]I}/2.91E+08 4.30E+07 15% 774% 5 Q286R/M298Q† Q143R/M156Q† {Gla swapFIX/Q44S}/ {Gla swap FIX/Q[44]S}/ 1.98E+08 2.75E+07 14% 529% 3Q286R/M298Q Q143R/M156Q {Gla swap FIX/M19K}/ {Gla swap FIX/M[19]K}/1.41E+08 5.22E+06 4% 375% 2 Q286R/M298Q Q143R/M156Q S52A/S60A/S[52]A/S[60]A/Q143R/M156Q 1.25E+08 1.14E+07 9% 333% 4 Q286R/M298Q Glaswap FIX/S52A/S60A/ Gla swap FIX/ 1.80E+08 1.81E+07 10% 480% 3Q286R/M298Q† S[52]A/S[60]A/Q143R/M156Q† {Gla swap {Gla swap 1.21E+087.07E+06 6% 322% 2 FIX/M19K/E40L/K43I/Q44S}/ FIX/M[19]K/E[40]L/K[43]I/Q286R/M298Q Q[44]}/Q286R/M298Q {Gla swap {Gla swap 2.71E+08 6.41E+07 24%720% 5 FIX/K43I}/T128N/P129A/ FIX/K[43]I}/T128N/P129A/ Q286R/M298Q†Q143R/M156Q† T239V T99V 4.64E+07 8.38E+06 18% 124% 2 T239I T99I 2.62E+076.51E+06 25% 70% 2 H257A/M298Q H117A/Q143R/M156Q 1.67E+07 4.27E+06 26%45% 5 S222A/H257A/Q286R/ S82A/H117A/Q143R/M156Q 1.65E+08 1.76E+07 11%440% 4 M298Q T128N/P129A/S222A/ T[128]N/P[129]A/S82A/H117A/ 1.55E+085.77E+07 37% 414% 9 H257A/Q286R/M298Q Q143R/M156Q T128N/P129A/S222A/T[128]N/P[129]A/S82A/H117A/ 1.73E+08 1.41E+07 8% 461% 2H257A/Q286R/M298Q† Q143R/M156Q† S52A/S60A/S222A/H257A/S[52]A/S[60]A/S82A/H117A/ 2.49E+08 8.78E+06 4% 665% 3 Q286R/M298QQ143R/M156Q H257S/Q286R/Q366V H117S/Q143R/Q217V 7.10E+07 3.16E+07 44%189% 11 S222A/H257A/Q286R/ S82A/H117A/Q143R/Q217V 1.00E+08 1.03E+07 10%268% 4 Q366V Q286R/M298Q/Q366N Q143R/M156Q/Q217N 1.17E+08 3.05E+07 26%312% 7 T128N/P129A/Q286R/M298Q/ T[129]N/P[129]A/Q143R/M156Q/ 1.42E+084.17E+07 29% 377% 3 Q366N† Q217N† {Gla swap {Gla swap 1.69E+08 3.89E+0723% 450% 5 FIX/K43I}/Q286R/M298Q/ FIX/K43I}/Q143R/M156Q/Q217N† Q366N†{Gla swap {Gla swap 2.52E+08 1.36E+07 5% 669% 2 FIX/K43I}/T128N/P129A/FIX/K43I}/T[128]N/P[129]A/ Q286R/M298Q/Q366N† Q143R/M156Q/Q217N†Q286R/H373F Q143R/H224F 9.01E+07 7.73E+06 9% 240% 2 T128N/P129A/T[128]N/P[129]A/Q143R/H224F 6.91E+07 2.15E+07 31% 184% 12 Q286R/H373FS52A/S60A/Q286R/H373F S[52]A/S[60]A/Q143R/H224F 9.44E+07 1.43E+07 15%252% 3 Q286R/M298Q/H373F Q143R/M156Q/H224F 1.36E+08 1.92E+07 14% 364% 5T128N/P129A/Q286R/M298Q/ T[128]N/P[129]A/Q143R/M156Q/ 1.33E+08 4.77E+0736% 354% 17 H373F H224F S52A/S60A/Q286R/M298Q/S[52]A/S[60]A/Q143R/M156Q/ 1.77E+08 3.63E+07 21% 472% 3 H373F H224FM298Q/H373F M156Q/H224F 7.21E+07 1.76E+07 24% 192% 4T128N/P129A/M298Q/H373F† T[128]N/P[129]A/M156Q/H224F† 6.07E+07 1.29E+0721% 161% 2 V158D/Q286R/E296V/M298Q V21D/Q143R/E154V/M156Q 1.49E+083.59E+07 24% 397% 11 S222A/T239V S82A/T99V 7.49E+07 2.57E+06 3% 200% 3Gla swap FIX/ Gla swap FIX/ 2.03E+08 3.16E+07 16% 541% 3S222A/T239V/Q286R S82A/T99V/Q143R Gla swap FIX/ Gla swap FIX/ 9.94E+071.83E+07 18% 264% 3 S222A/T239V/Q286R† S82A/T99V/Q143R†T239V/Q286R/M298Q T99V/Q143R/M156Q 1.72E+08 4.92E+07 29% 459% 5 Gla swapFIX/ Gla swap FIX/ 2.53E+08 4.78E+07 19% 675% 3 T239V/Q286R/M298QT99V/Q143R/M156Q Gla swap FIX/ Gla swap FIX/ 1.79E+08 3.81E+07 21% 477%4 T239V/Q286R/M298Q† T99V/Q143R/M156Q† T128N/P129A/T239V/Q286R/T[128]N/P[129]A/T99V/Q143R/ 1.04E+08 2.43E+07 23% 276% 4 M298Q† M156Q†S222A/T239V/H257A/ S82A/T99V/H117A/Q143R/ 2.14E+08 4.48E+07 21% 571% 5Q286R/M298Q M156Q T128N/P129A/S222A/T239V/ T[128]N/P[129]A/S82A/T99V/1.21E+08 5.58E+06 5% 323% 3 H257A/ H117A/Q143R/M156Q† Q286R/M298Q†T239V/Q286R/H373F T99V/Q143R/H224F 1.06E+08 1.34E+07 13% 283% 2T239V/Q286R/M298Q/H373F T99V/Q143R/M156Q/H224F 1.70E+08 1.13E+07 7% 454%2 T128N/P129A/T239V/Q286R/ T[128]N/P[129]A/T99V/Q143R/ 2.36E+08 2.77E+0712% 627% 3 M298Q/H373F† M156Q/H224F† V158D/T239I/E296V/M298QV21D/T99I/E154V/M156Q 1.45E+08 1.18E+07 8% 387% 4 T239I/Q286R T99I/Q143R5.79E+07 1.39E+07 24% 155% 3 S222A/T239I S82A/T99I 3.05E+07 9.26E+06 30%81% 4 GlaSwapFIX/S222A/T239I/ Gla swap FIX/ 6.77E+07 4.44E+06 7% 181% 2Q286R S82A/T99I/Q143R T239I/Q286R/M298Q T99I/Q143R/M156Q 1.13E+083.68E+06 3% 301% 2 Gla swap FIX/ Gla swap FIX/ 1.25E+08 2.13E+07 17%334% 2 T239I/Q286R/M298Q T99I/Q143R/M156Q T128N/P129A/T239I/Q286R/T[128]N/P[129]A/T99I/Q143R/ 8.17E+07 8.17E+06 10% 217% 3 M298Q† M156Q†S222A/T239I/H257A/Q286R/ S82A/T99I/H117A/Q143R/M156Q 1.14E+08 2.22E+0719% 304% 3 M298Q T239I/Q286R/H373F T99I/Q143R/H224F 6.18E+07 9.27E+0615% 165% 3 V158D/T239V/E296V/M298Q V21D/T99V/E154V/M156Q 2.22E+081.39E+07 6% 591% 2 V158D/T239V/E296V/M298Q† V21D/T99V/E154V/M156Q†1.65E+08 2.12E+06 1% 438% 2 T239V/Q286R T99V/Q143R 8.84E+07 7.16E+05 1%236% 2 T239I/Q286R/M298Q/ T99I/Q143R/M156Q/H224F 1.08E+08 2.32E+07 21%289% 7 H237F T128N/P129A/T239I/Q286R/ T[128]N/P[129]A/T99I/Q143R/1.30E+08 2.51E+07 19% 345% 5 M298Q/H237F† M156Q/H224F† H257S/Q286R/M298QH117S/Q143R/M156Q 1.40E+08 8.97E+06 6% 372% 4 Gla swap FIX/ Gla swapFIX/ 8.53E+07 1.66E+07 20% 227% 3 Q286R/S222A/H257S Q143R/S82A/H117SS222A/H257S/Q286R/ S82A/H117S/Q143R/M156Q 1.58E+08 1.76E+07 11% 420% 2M298Q H257S/Q286R/M298Q/ H117S/Q143R/M156Q/H224F 1.52E+08 3.35E+07 22%407% 7 H373F S222A/Q286R/M298Q/ S82A/Q143R/M156Q/H224F 1.48E+08 2.23E+062% 395% 2 H373F Gla swap FIX/S222A/ Gla swap FIX 2.84E+08 4.85E+07 17%758% 3 Q286R/M298Q/H373F S82A/Q143R/M156Q/H224F S222A/Q286R/M298QS82A/Q143R/M156Q 1.29E+08 1.86E+07 14% 343% 3 Gla swap FIX/ Gla swap FIX2.10E+08 4.28E+07 20% 559% 5 S222A/Q286R/M298Q S82A/Q143R/M156QT128N/P129A/A175S/ T[128]N/P[129]A/A39S/Q217V 3.38E+07 3.06E+06 9% 90% 2Q366V A122N/G124S/A175S/ A[122]N/G[124]S/A39S/Q217V 3.02E+07 7.05E+0623% 80% 5 Q366V T128N/P129A/A175S/ T[128]N/P[129]A/A39S/S82A 1.72E+073.18E+06 18% 46% 3 S222A A122N/G124S/A175S/ A[122]N/G[124]S/A39S/S82A2.08E+07 5.05E+06 24% 56% 5 S222A T128N/P129A/A175S/T[128]N/P[129]A/A39S/Q143R 3.33E+07 1.46E+06 4% 89% 3 Q286RA122N/G124S/A175S/ A[122]N/G[124]S/A39S/Q143R 4.11E+07 5.27E+06 13% 110%5 Q286R Gla swap FIX/ Gla swap FIX/ 1.22E+08 3.17E+07 26% 327% 8S222A/Q286R/H373F S82A/Q143R/H224F V158D/E296V/M298Q/V21D/E154V/M156Q/H224F 1.51E+08 8.39E+06 6% 402% 3 H373FH257A/Q286R/M298Q H117A/Q143R/M156Q 1.13E+08 1.55E+07 14% 301% 3 Glaswap FIX/ Gla swap FIX/ 3.88E+07 2.74E+06 7% 104% 3 T128N/P129A/A175S/T[128]N/P[129]A/A39S/S82A/ S222A/Q286R Q143R Gla swap FIX/ Gla swap FIX/4.13E+07 8.99E+06 22% 110% 6 A122N/G124S/A175S/A[122]N/G[124]S/A39S/S82A/ S222A/Q286R Q143R T128N/P129A/A175S/T[128]N/P[129]A/A39S/Q143R/ 7.21E+07 1.14E+07 16% 192% 3 Q286R/M298QM156Q A122N/G124S/A175S/ A[122]N/G[124]S/A39S/Q143R/ 7.43E+07 1.10E+0715% 198% 3 Q286R/M298Q M156Q T128N/P129A/A175S/T[128]N/P[129]A/A39S/S82A/ 6.89E+07 3.36E+06 5% 184% 3S222A/H257A/Q286R/ H117A/Q143R/M156Q M298Q A122N/G124S/A175S/A[122]N/G[124]S/A39S/S82A/ 8.40E+07 5.72E+06 7% 224% 3S222A/H257A/Q286R/ H117A/Q143R/M156Q M298Q T128N/P129A/A175S/T[128]N/P[129]A/A39S/Q143R/ 5.72E+07 3.36E+06 6% 153% 3Q286R/M298Q/H373F M156Q/H224F A122N/G124S/A175S/A[122]N/G[124]S/A39S/Q143R/ 8.39E+07 9.99E+06 12% 224% 3Q286R/M298Q/H373F M156Q/H224F V158D/Q286R/E296V/M298Q/V21D/Q143R/E154V/M156Q/ 2.39E+08 3.82E+07 16% 638% 5 H373F H224FM298Q/Q366N/H373F† M156Q/Q217N/H224F† 7.05E+07 1.78E+07 25% 188% 3T239V/M298Q/H373F† T99V/M156Q/H224F† 4.43E+07 1.10E+07 25% 118% 3T239I/M298Q/H373F† T99I/M156Q/H224F† 3.47E+07 4.57E+06 13% 92% 3T128N/P129A/Q286R/M298Q/ T[128]N/P[129]A/Q143R/M156Q/ 1.33E+08 1.81E+0714% 355% 2 Q366N/H373F† Q217N/H224F† T239V/Q286R/M298Q/Q366N†T99V/Q143R/M156Q/Q217N† 1.85E+08 5.96E+07 32% 491% 4T239I/Q286R/M298Q/Q366N† T99I/Q143R/M156Q/Q217N† 7.40E+07 1.40E+07 19%197% 4 TF-independent assay WT (NovoSeven ®) WT (NovoSeven ®) 9.8 3.030% 88% 14 WT (NovoSeven-RT ®) WT (NovoSeven-RT ®) 12.4 4.3 35% 112% 12WT WT 11.1 2.7 25% 100% 5 WT† WT† 6.9 2.2 31% 100% 7 T128N/P129AT[128]N/P[129]A 17.0 5.2 31% 153% 3 Gla swap FIX Gla swap FIX 41.3 3.07% 373% 2 A122N/G124S A[122]N/G[124]S 3.4 0.6 16% 31% 2 S52A/S60AS[52]A/S[60]A 3.8 34% 1 M298Q† M156Q† 69.9 49.4 71% 1013% 3T128N/P129A/M156Q† T[128]N/P[129]A/M156Q† 90.8 70.8 78% 1316% 5V158D/E296V/M298Q V21D/E154V/M156Q 1221.7 307.0 25% 11025% 4V158D/E296V/M298Q† V21D/E154V/M156Q† 984.5 308.4 31% 14265% 2T128N/P129A/V158D/E296V/ T[128]N/P[129]A/V21D/E154V/ 1375.8 140.3 10%12415% 3 M298Q M156Q S52A/S60A/V158D/E296V/ S[52]A/S[60]A/V21D/E154V/1760.1 575.0 33% 15883% 3 M1298Q M156Q Q286R Q143R 10.3 0.7 7% 93% 3T128N/P129A/Q286R T[128]N/P[129]A/Q143R 8.7 4.3 50% 78% 5T128N/P129A/Q286R† T[128]N/P[129]A/Q143R† 10.5 5.6 53% 152% 6S52A/S60A/S82A S[52]A/S[60]A/S82A 10.2 4.7 47% 92% 3 T128N/P129A/S222AT[128]N/P[129]A/S82A 4.8 43% 1 S52A/S60A/S222A S[52]A/S[60]A/S82A 19.6177% 1 H257S H117S 3.1 1.1 35% 28% 7 Q366V Q217V 4.3 0.6 14% 39% 2 GlaswapFIX/Q366V Gla swapFIX/Q217V 90.0 17.7 20% 812% 2 Q286R/H257AQ143R/H117A 4.3 2.1 49% 39% 3 Q286R/H257A† Q143R/H117A† 2.5 0.0 0% 36% 2Gla swap FIX/ Gla swap FIX/ 15.5 2.9 18% 140% 4 T128N/P129A/T[128]N/P[129]A/ S222A/Q286R S82A/Q143R Gla swap FIX/ Gla swap FIX/ 21.36.5 31% 309% 5 T128N/P129A/ T[128]N/P[129]A/ S222A/Q286R† S82A/Q143R†Gla swap FIX/ Gla swap FIX/ 2.9 26% 1 S52A/S60A/S222A/Q286RS[52]A/S[60]A/S82A/Q143R Q286R/S222A/H257A Q143R/S82A/H117A 21.3 6.5 31%193% 5 Q286R/M298Q Q143R/M156Q 79.9 18.9 24% 721% 5 Q286R/M298Q†Q143R/M156Q† 162.4 79.9 49% 2353% 12 Q286R/M298Q§ Q143R/M156Q§ 135.1 7.35% 1957% 2 Gla swap FIX/ Gla swap FIX/ 672.7 79.1 12% 6070% 4Q286R/M298Q Q143R/M156Q Gla swap FIX/ Gla swap FIX/ 678.2 249.0 37%9826% 11 Q286R/M298Q† Q143R/M156Q† T128N/P129A/Q286R/T[128]N/P[129]A/Q143R/ 81.6 13.4 16% 736% 4 M298Q M156QT128N/P129A/Q286R/ T[128]N/P[129]A/Q143R/ 212.5 135.4 64% 3079% 10M298Q† M156Q† Gla swap FIX/ Gla swap FIX/ 83.8 35.3 42% 756% 6T128N/P129A/Q286R/ T[128]N/P[129]A/Q143R/ M298Q M156Q Gla swap FIX/ Glaswap FIX/ 751.9 305.3 41% 10895% 6 T128N/P129A/Q286R/T[128]N/P[129]A/Q143R/ M298Q† M156Q† {Gla swap FIX/E40L}/ {Gla swapFIX/E[40]L}/ 814.1 89.0 11% 7346% 2 Q286R/M298Q Q143R/M156Q {Gla swapFIX/K43I}/ {Gla swap FIX/K[43]I}/ 902.4 360.6 40% 8144% 11 Q286R/M298QQ143R/M156Q {Gla swap FIX/K43I}/ {Gla swap FIX/K[43]I}/ 794.2 178.7 23%11508% 6 Q286R/M298Q† Q143R/M156Q† {Gla swap FIX/Q44S}/ {Gla swapFIX/Q[44]S}/ 729.0 4.5 1% 6578% 2 Q286R/M298Q Q143R/M156Q {Gla swapFIX/M19K}/ {Gla swap FIX/M[19]K}/ 512.0 51.4 10% 4620% 2 Q286R/M298QQ143R/M156Q S52A/S60A/ S[52]A/S[60]A/Q143R/M156Q 216.8 1.6 1% 1956% 2Q286R/M298Q Gla swap FIX/S52A/S60A/ Gla swap FIX/S[52]A/ 988.7 207.5 21%14327% 2 Q286R/M298Q S[60]A/Q143R/M156Q {Gla swap FIX/K43I}/ {Gla swapFIX/K[43]I}/ 389.4 34.3 9% 5642% 2 T128N/P129A/Q286R/M298Q†T[128]N/P[129]A/ Q143R/M156Q† S222A/H257A/Q286R/ S82A/H117A/Q143R/M156Q345.3 99.9 29% 3116% 3 M298Q T128N/P129A/S222A/T[128]N/P[129]A/S82A/H117A/ 24.8 17.2 69% 224% 4 H257A/Q286R/M298QQ143R/M156Q T128N/P129A/S222A/ T[128]N/P[129]A/S82A/H117A/ 82.6 40.2 49%1196% 3 H257A/Q286R/M298Q† Q143R/M156Q† S52A/S60A/S222A/H257A/S[52]A/S[60]A/S82A/H117A/ 115.6 62.9 54% 1043% 2 Q286R/M298Q Q143R/M156QH257S/Q286R/Q366V H117S/Q143R/Q217V 7.7 1.8 23% 69% 2S222A/H257A/Q286R/Q366V S82A/H117A/Q143R/Q217V 12.5 2.8 23% 113% 2Q286R/M298Q/Q366N Q143R/M156Q/Q217N 65.9 33.4 51% 595% 5T129N/P129A/Q286R/M298Q/ T[129]N/P[129]A/Q143R/M156Q/ 64.6 28.7 44% 936%4 Q366N† Q217N† {Gla swap FIX/K43I}/ {Gla swap FIX/K[43]I}/ 84.9 76.590% 1230% 4 Q286R/M298Q/Q217N† Q143R/M156Q/Q217N† {Gla swap FIX/K43I}/{Gla swap FIX/K[43]I}/ 218.5 137.8 63% 3166% 3 T128N/P129A/Q286R/M298Q/T[128]N/P[129]A/Q143R/M156Q/ Q366N† Q217N† Q286R/H373F Q143R/H224F 81.6123.7 152% 736% 9 T128N/P129A/ T[128]N/P[129]A/Q143R/H224F 6.6 0.9 13%59% 2 Q286R/H373F S52A/S60A/Q286R/H373F S[52]A/S[60]A/Q143R/H224F 30.1272% 1 Q286R/M298Q/H373F Q143R/M156Q/H224F 114.8 24.7 22% 1036% 5T128N/P129A/Q286R/M298Q/ T[128]N/P[129]A/Q143R/M156Q/ 30.7 8.9 29% 277%4 H373F† H224F† S52A/S60A/Q286R/M298Q/ S[52]A/S[60]A/Q143R/M156Q/ 63.310.8 17% 571% 3 H373F H224F M298Q/H373F M156Q/H224F 96.4 47.0 49% 870% 5T128N/P129A/M298Q/H373F T[128]N/P[129]A/M156Q/H224F 91.6 48.0 52% 1327%3 V158D/Q286R/E296V/M298Q V21D/Q143R/E154V/M156Q 1023.9 339.3 33% 9240%5 S222A/T239V S82A/T99V 3.0 27% 1 Gla swap FIX/ Gla swap FIX/ 17.4 2.213% 157% 3 S222A/T239V/Q286R S82A/T99V/Q143R Gla swap FIX/ Gla swap FIX/87.9 61.7 70% 1274% 4 S222A/T239V/Q286R† S82A/T99V/Q143R†T239V/Q286R/M298Q T99V/Q143R/M156Q 29.3 6.2 21% 264% 4 Gla swap FIX/ Glaswap FIX/ 277.7 64.2 23% 2506% 3 T239V/Q286R/M298Q T99V/Q143R/M156Q Glaswap FIX/ Gla swap FIX/ 902.4 323.5 36% 13076% 6 T239V/Q286R/M298Q†T99V/Q143R/M156Q† T128N/P129A/ T[128]N/P[129]A/ 229.7 134.1 58% 3329% 5T239V/Q286R/M298Q† T99V/Q143R/M156Q† S222A/T239V/H257A/S82A/T99V/H117A/Q143R/ 143.0 93.1 65% 1290% 10 Q286R/M298Q M156QT128N/P129A/ T[128]N/P[129]A/ 179.0 80.5 45% 2593% 5 S222A/T239V/H257A/S82A/T99V/H117A/Q143R/ Q286R/M298Q M156Q T239V/Q286R/H373FT99V/Q143R/H224F 12.2 110% 1 T239V/Q286R/M298Q/H373FT99V/Q143R/M156Q/H224F 40.7 5.2 13% 367% 2 T128N/P129A/T99V/Q143R/T[128N]/P129]A/T99V/Q143R/ 290.0 72.9 25% 4203% 4 M156Q/H224FM156Q/H224F V158D/T239I/E296V/M298Q V21D/T99I/E154V/M156Q 216.3 32.5 15%1951% 2 T239I/Q286R T99I/Q143R 4.6 1.3 28% 41% 4 S222A/T239I S82A/T99I1.7 15% 1 Gla swap FIX/ Gla swap FIX/ 20.3 184% 1 S222A/T239I/Q286RS82A/T99I/Q143R T239I/Q286R/M298Q T99I/Q143R/M156Q 11.3 4.0 35% 102% 4Gla swap FIX/ Gla swap FIX/ 244.0 9.6 4% 2202% 2 T239I/Q286R/M298QT99I/Q143R/M156Q T128N/P129A/T239I/Q286R/ T[128N]/P129]A/T99I/Q143R/77.8 40.3 52% 1128% 5 M298Q M156Q S222A/T239I/H257A/Q286R/S82A/T99I/H117A/Q143R/M156Q 51.6 5.7 11% 466% 2 M298QV158D/T239V/E296V/M298Q V21D/T99V/E154V/M156Q 1864.3 374.0 20% 16823% 2V158D/T239V/E296V/M298Q† V21D/T99V/E154V/M156Q† 4231.6 913.4 22% 61315%4 T239V/Q286R T99V/Q143R 11.8 4.1 35% 106% 4 T239I/Q286R/M298Q/H373FT99I/Q143R/M156Q/H224F 13.1 3.8 29% 118% 3 T128N/P129A/T239I/Q286R/T[128]N/P[129]A/T99I/Q143R 113.3 43.7 39% 1642% 5 M298Q/H373F†M156Q/H224F† H257S/Q286R/M298Q H117S/Q143R/M156Q 27.4 4.1 15% 247% 4 Glaswap FIX/ Gla swap FIX/ 20.5 3.6 18% 185% 2 S8222A/H257S/Q143RS82A/H117S/Q143R S222A/Q286R/M298Q/ S82A/Q143R/M156Q/H224F 41.7 9.1 22%376% 4 H373F H257S/Q286R/M298Q/H373F H117S/Q143R/M156Q/H224F 30.4 9.130% 274% 3 S82A/Q143R/M156Q/H224F S82A/Q143R/M156Q/H224F 430.2 126.8 29%3883% 3 Gla swap FIX/S222A/ Gla swap FIX/ 192.1 36.8 19% 1733% 2Q286R/M298Q/H373F S82A/Q143R/M156Q/H224F S222A/Q286R/M298QS82A/Q143R/M156Q 252.9 7.4 3% 2282% 2 Gla swap FIX/ Gla swap FIX/ 414.781.3 20% 3742% 2 S222A/Q286R/M298Q S82A/Q143R/M156Q T128N/P129A/A175S/T[128]N/P[129]A/A39S/Q217V 3.4 1.0 29% 30% 2 Q366V A122N/G124S/A175S/A[122]N/G[124]S/A39S/Q217V 3.0 0.8 26% 27% 4 Q366V T128N/P129A/A175S/T[128]N/P[129]A/A39S/S82A 1.9 0.5 26% 17% 2 S222A T128N/P129A/A175S/T[128]N/P[129]A/A39S/Q143R 3.3 1.2 37% 29% 4 Q286R A122N/G124S/A175S/A[122]N/G[124]S/A39S/Q143R 3.0 0.7 23% 27% 2 Q286R Gla swap FIX/ Glaswap FIX/ 81.2 66.7 82% 732% 2 S222A/Q286R/H373F S82A/Q143R/H224FV158D/E296V/M298Q/H373F V21D/E154V/M156Q/H224F 1297.2 486.1 37% 11706% 4H257A/Q286R/M298Q H117A/Q143R/M156Q 61.5 43.8 71% 555% 2 Gla swap FIX/Gla swap FIX/ 30.5 276% 1 T128N/P129A/A175S/S222A/T[128]N/P[129]A/A39S/S82A/ Q286R Q143R T128N/P129A/A175S/T[128]N/P[129]A/A39S/S82A/ 20.3 3.8 19% 183% 2 S222A/H257A/Q286R/M298QH117A/Q143R/M156Q V158D/Q286R/E296V/M298Q/ V21D/Q143R/E154V/M156Q/ 573.6100.4 18% 5176% 6 H373F H224F M298Q/Q366N/H373F† M156Q/Q217N/H224F†125.9 75.2 60% 1825% 4 T239V/M298Q/H373F† T99V/M156Q/H224F† 319.5 125.039% 4629% 6 T239I/M298Q/H373F† T99I/M156Q/H224F† 138.2 101.9 74% 2003% 7T128N/P129A/Q286R/M298Q/ T[128]N/P[129]A/Q143R/M156Q/ 160.9 43.3 27%2331% 4 Q366N/H373F† Q217N/H224F† T239V/Q286R/M298Q/Q366N†T99V/Q143R/M156Q/Q2176N† 64.2 36.3 57% 931% 3 T2391/Q286R/M298Q/Q366N†T99I/Q143R/M156Q/Q217N† 88.8 23.5 26% 1287% 5 †produced in CHOX cells§produced in CHOX stable cell line clone 52-5F7

Example 5 Determination of the Inhibition of FVIIa/TF or FVIIa byAT-III/Heparin

The potency of the interaction between the AT-III/heparin complex andFVIIa in the presence or absence of soluble tissue factor (sTF), i.e.TF-dependent or TF-independent, was assessed by measuring the level ofinhibition of various concentrations of AT-III on the catalytic activityof FVIIa/sTF towards a substrate, Mesyl-FPR-ACC. The K_(0.5) value wasdetermined for each FVIIa variant tested, which corresponds to the molarconcentration of AT-III that was required for 50% inhibition (IC₅₀) ofFVIIa variant in a 30 minute assay at room temperature (˜250).

Two separate assays were prepared, one with sTF and one without sTF. A 2μM solution of AT-III/heparin (final 5 μM heparin) was prepared bymixing 26.4 μL of 151.7 μM AT-III (plasma purified human AT-III;Molecular Innovations) with 50 μL of 0.2 mM LMW heparin (CalBiochem),400 μL of 5× assay buffer (100 mM Hepes, 750 mM NaCl, 25 mM CaCl₂, 0.05%BSA, 0.5% PEG 8000, pH 7.4) and 1.523 mL of reagent grade water. Thissolution was for use as the highest concentration in the TF-dependentassay. A solution containing 4 μM AT-III/heparin (final 5 μM heparin)was prepared for use in the TF-independent assay by mixing 52.8 μL of151.7 μM AT-III (Molecular Innovations) with 50 μL of 0.2 mM LMW heparin(CalBiochem), 400 μL of 5× assay buffer and 1.497 mL of reagent gradewater. The AT-III/heparin solutions were incubated for 5-10 minutes atroom temperature and then diluted two-fold down in a 96 deep-wellpolypropylene plate with a final volume of 1 mL containing 5 μM heparin,resulting in dilutions of 2000, 1000, 500, 250, 125, 62.5, 31.25 and 0nM, or 4000, 2000, 1000, 500, 250, 125, 62.5, and 0 nM. The FVIIavariants and wild-type FVIIa were diluted to 250 nM in 1× assay buffer(20 mM Hepes, 150 mM NaCl, 5 mM CaCl₂, 0.01% BSA, 0.1% PEG 8000, pH7.4). For the TF-dependent assay, 5 nM FVIIa/50 nM sTF complexes wereformed by mixing 20 μL of FVIIa with 10 μL of 5 μM sTF (R&D SystemsHuman Coagulation Factor III: #2339-PA), 200 μL 5× assay buffer and 770μL reagent grade water and incubating the solutions for 10-15 minutes atroom temperature. For the TF-independent assay, 100 μL of FVIIa wasmixed with 200 μL 5× assay buffer and 700 μL reagent grade water toproduce 25 nM solutions of FVIIa. To start the assay, 25 μL of theFVIIa/TF or FVIIa alone solutions were separately mixed with 25 μL ofeach dilution of AT-III/heparin in wells of a 96-well black half areaassay plate (Nunc). The final assay conditions for the TF-dependentassay were 2.5 nM FVIIa/25 nM sTF and AT-III/heparin concentrationsranging from 1000 nM to 0 nM. For the TF-independent assay, FVIIaconcentrations were 12.5 nM FVIIa and AT-III/heparin concentrationsranged from 2000 nM to 0 nM. The plates were incubated for 30 minuteswith shaking at room temperature (˜25° C.).

A stock solution of FVIIa substrate (Mesyl-FPR-ACC) was prepared bydissolving the substrate in DMSO to 20 mM then preparing a workingsolution of 0.5 mM in 1× assay buffer. Following incubation of the assayplate from above, 50 μl of the FVIIa substrate was added to each well ofthe assay plate. The reactions were mixed and the residual activity ofFVIIa was assessed by following the initial rates of substrate cleavagefor 15 minutes in a fluorescence reader set to 30° C.

To determine the degree of inhibition by AT-III/heparin for FVIIa orFVIIa variants, raw data collected with the SoftMax Pro application(Molecular Devices) were exported as .XML files. Further non-linear dataanalyses were performed with XLfit4, a software package for automatedcurve fitting and statistical analysis within the Microsoft Excelspreadsheet environment (IDBS Software). The spreadsheet template wasused to calculate the AT-III dilution series, ratio of AT-III to FVIIa,and the Vi/Vo ratios for each FVIIa replicate at each experimentalAT-III concentration. Non-linear regression analyses of residual FVIIaactivity (expressed as Vi/Vo) versus AT-III concentration was processedusing XLfit4 and a hyperbolic inhibition equation of the form((C+(Amp*(1−(X/(K_(0.5)+X))))); where C=the offset (fixed at 0 to permitextrapolation of data sets that do not reach 100% inhibition during thecourse of the assay), Amp=the amplitude of the fit and K_(0.5), whichcorresponds to the concentration of AT-III required for half-maximalinhibition under the assay conditions. For several FVIIa variants,AT-III inhibited less than 20-25% of the of the total protease activityat the highest tested concentration of AT-III, representing an upperlimit of detection for the assay. Variants with less than 20-25% maximalinhibition were therefore assigned a lower limit K_(0.5) value (5 μM forTF-dependent and 10 μM for TF-independent) and in most cases areexpected to have AT-III resistances greater than the reported value.

Tables 16 and 17 provide the results of the assays that were performedusing FVIIa variants expressed in Freestyle TM 293-F cells and/or BHK-21cells, in the presence and absence of TF, respectively. The results arepresented both as the fitted K0.5 parameter and as a representation ofthe extent of AT-III resistance for each variant compared to thewild-type FVIIa expressed as a ratio of their fitted K_(0.5) values(K_(0.5) variant/K_(0.5) wild-type). Several FVIIa variants exhibitedincreased resistance to AT-III compared to wild-type FVIIa. For example,Q286R-FVIIa (i.e. FVIIa containing the Q286R mutation),Q286R/S222A-FVIIa, Q286R/S222A/Gla Swap FIX-FVIIa,A175S/Q286R/Q366V-FVIIa, Q286M-FVIIa, Q286L-FVIIa and Q286Y-FVIIa areamong the group which exhibited resistance to AT-III in the absence ofTF that was over 4 times greater than that of wild-type FVIIa.

TABLE 16 Inhibition of FVIIa variants by AT-III/heparin in the presenceof TF TE-Dependent ATIII Resistance Assay Mutation (mature FVII Mutation(Chymotrypsin 293-F Cells BHK-21 Cells numbering) Numbering) K_(0.5)(nM) K_(0.5 mut)/K_(0.5 wt) K_(0.5) (nM) K_(0.5 mut)/K_(0.5 wt) WT WT72.3 1.0 56.0 1.0 V158D/E296V/M298Q V21D/E154V/M156Q 75.1 1.0 79.0 1.4Q286R Q143R 60.6 0.8 59.1 1.1 S222A S82A 47.6 0.7 43.9 0.8 H257S H117S50.6 0.7 52.9 0.9 H373D H224D 423.6 5.9 H373E H224E 152.1 2.1 H373SH224S 64.2 0.9 H373F H224F 38.7 0.5 H373A H224A 76.9 1.1 Q366D Q217D2239.2 31.0 Q366E Q217E 116.2 1.6 Q366N Q217N 75.3 1.0 Q366T Q217T 57.50.8 Q366S Q217S 107.2 1.5 Q366V Q217V 25.8 0.4 20.0 0.4 A175S A39S 112.41.6 A122N/G124S A[122]N/G[124]S 48.2 0.7 Q286R/S222A Q143R/S82A 53.3 1.0Q286R/S222A/Gla Q143R/S82A/Gla swap 83.7 1.2 Swap FIX FIX Q286R/M298QQ143R/M156Q 74.2 1.3 Q286R/M298Q/K341Q Q143R/M156Q/K192Q 21.8 0.4Q286R/M298Q/K199E Q143R/M156Q/K60cE 101.1 1.8 P321K P170iK 97.5 1.7P321E P170iE 66.0 1.2 P321Y P170iY 49.5 0.9 P321S P170iS 60.7 1.1 T239ST99S 254.6 3.5 T239Q T99Q 117.2 2.1 T239V T99V 42.5 0.8 T239L T99L 81.11.4 T239H T99H 52.0 0.9 T239I T99I 125.3 2.2 H257A/M298Q H117A/M156Q89.1 1.6 S222A/H257A/Q286R/ S82A/H117A/Q143R/M156Q 66.6 0.9 M298QQ286R/Q366V Q143R/Q217V 62.0 1.1 A175S/Q286R/Q366V A39S/Q143R/Q217V 72.01.3 S222A/Q286R/Q366V S82A/Q143R/Q217V 38.5 0.7 Q286M Q143M 53.1 0.7Q286L Q143L 114.4 1.6 Q286Y Q143Y 131.3 1.8 Q366I Q217I 23.2 0.3 Q366LQ217L 23.0 0.3 Q366M Q217M 35.4 0.5

TABLE 17 Inhibition of FVIIa variants by AT-III/heparin in the absenceof TF TF-Independent ATIII Resistance Assay Mutation 293-F Cells BHK-21Cells Mutation (mature (Chymotrypsin K_(0.5) K_(0.5) FVII numbering)Numbering) (nM) K_(0.5 mut)/K_(0.5 wt) (nM) K_(0.5 mut)/K_(0.5 wt) WT WT2265 1.0 2222 1.0 V158D/E296V/M298Q V21D/E154V/M156Q 389 0.2 415 0.2Q286R Q143R 10000 >4.4 10000 >4.5 S222A S82A 2338 1.0 2088 0.9 H257SH117S 5884 2.6 5584 2.5 H373D H224D 10000 >4.4 H373E H224E 6949 3.1H373S H224S 9513 4.2 H373F H224F 1306 0.6 H373A H224A 10000 >4.4 Q366DQ217D 10000 >4.4 Q366E Q217E 6901 3.0 Q366N Q217N 5186 2.3 Q366T Q217T5885 2.6 Q366S Q217S 10000 >4.4 Q366V Q217V 487 0.2 531 0.2 A175S A39S5785 2.6 A122N/G124S A[122]N/G[124]S 2926 1.3 Q286R/S222A Q143R/S82A10000 >4.5 Q286R/S222A/Gla Q143R/S82A/Gla swap 10000 >4.4 Swap FIX FIXQ286R/M298Q Q143R/M156Q 3663 1.6 Q286R/M298Q/K341Q Q143R/M156Q/K192Q 72Q286R/M298Q/K199E Q143R/M156Q/K60cE 5182 2.3 P321K P170iK 5274 2.4 P321EP170iE 3666 1.6 P321Y P170iY 705 0.3 P321S P170iS 2689 1.2 T239S T99S10000 >4.4 T239Q T99Q 2222 1.0 T239V T99V 2644 1.2 T239L T99L 8532 3.8T239H T99H 10000 >4.5 T239I T99I 10000 >4.5 H257A/M298Q H117A/M156Q 13440.6 S222A/H257A/Q286R/ S82A/H117A/Q143R/ 7742 3.4 M298Q M156QQ286R/Q366V Q143R/Q217V 2251 1.0 A175S/Q286R/Q366V A39S/Q143R/Q217V10000 >4.5 S222A/Q286R/Q366V S82A/Q143R/Q217V 3398 1.5 Q286M Q143M10000 >4.4 Q286L Q143L 10000 >4.4 Q286Y Q143Y 10000 >4.4 Q366I Q217I 5990.3 Q366L Q217L 1708 0.8 Q366M Q217M 914 0.4

A further set of experiments were performed to assess the inhibition ofFVIIa variants by AT-III/heparin in the absence of TF using the sameassay as described above with minor modifications. Full-length,unfractionated heparin (Calbiochem) was used instead of low molecularweight heparin (LMW-heparin) to increase the rate of the inhibitionreaction (see e.g., Olson et al. (2004) Thromb Haemost 92(5), 929-939).The incubation time of the assay was increased to 60 minutes, and theconcentration of mesyl-FPR-ACC substrate used to ascertain residualactivity was increased to a final concentration of 0.5 mM.

Table 18 provides the results of the assays that were performed in theabsence of TF using FVIIa variants expressed in BHK-21 cells and CHOXcells. The results are presented both as the fitted K_(0.5) parameterand as a representation of the extent of AT-III resistance for eachvariant compared to the wild-type FVIIa expressed as a ratio of theirfitted K_(0.5) values (K_(0.5) mutant/K_(0.5) wild-type). The standarddeviation (SD) and number of assays (n) also are shown.

TABLE 18 Inhibition of FVIIa variants by AT-III/heparin in the absenceof TF Mutation (mature FVII Mutation (Chymotrypsin K_(0.5) K_(0.5 mut)/numbering) Numbering) (nM) SD % CV K_(0.5 wt) n WT (NovoSeven ®) WT(NovoSeven ®) 424.3 70.9 17% 1.08 33 WT (NovoSeven-RT ®) WT(NovoSevenRT ®) 424.2 60.5 14% 1.08 5 WT WT 393.8 67.8 17% 1.00 4 WT†WT† 503.0 120.0 24% 1.00 4 T128N/P129A T[128]N/P[129]A 465.3 28.1 6%1.18 2 Gla swap FIX Gla swap FIX 298.9 0.76 1 K109N K[109]N 330.1 72.322% 0.84 2 A122N/G124S A[122]N/G[124]S 372.5 28.6 8% 0.95 2 S52A/S60AS[52]A/S[60]A 360.6 0.92 1 M298Q M156Q 120.1 14.1 12% 0.31 5 M298Q†M156Q† 130.0 14.3 11% 0.26 2 T128N/P129A/M298Q† T[128]N/P[129]A/M156Q†143.9 14.5 10% 0.29 2 V158D/E296V/M298Q V21D/E154V/M156Q 75.5 10.1 13%0.19 27 V158D/E296V/M298Q† V21D/E154V/M156Q† 77.4 18.0 23% 0.15 7T128N/P129A/V158D/E296V/ T[128]N/P[129]A/V21D/E154V/ 81.6 3.8 5% 0.21 2M298Q M156Q S52A/S60A/V158D/E296V/M1298Q S[52]A/S[60]A/V21D/E154V/ 78.82.9 4% 0.20 2 M156Q Q286R Q143R 1085.1 320.0 29% 2.76 20T128N/P129A/Q286R T[128]N/P[129]A/Q143R 1645.2 440.2 27% 4.18 9T128N/P129A/Q286R† T[128]N/P[129]A/Q143R† 1739.2 467.0 27% 3.46 5S52A/S60A/Q143R S[52]A/S[60]A/Q143R 1318.0 376.8 29% 3.35 2 S222A S82A383.5 84.4 22% 0.97 3 T128N/P129A/S222A T[128]N/P[129]A/S82A 401.0 1.021 H257S H117S 722.8 1.84 1 Q366V Q217V 101.1 24.7 24% 0.26 3 GlaswapFIX/Q366V Gla swapFIX/Q217V 108.2 5.8 5% 0.27 2 A175S A39S 1328.096.2 7% 3.37 3 K109N/A175S K[109]N/A39S 2031.8 401.2 20% 5.16 2S119N/L121S/A175S S[119]N/L[121]S/A39S 1637.2 171.3 10% 4.16 2T128N/P129A/A175S T[128]N/P[129]A/A39S 1392.7 295.3 21% 3.54 2A122N/G124S/A175S A[122]N/G[124]S/A39S 1345.8 241.1 18% 3.42 2Q286R/H257A Q143R/H117A 2398.7 551.2 23% 6.09 9 Q286R/H257A†Q143R/H117A† 2800.8 938.4 34% 5.57 5 Q286R/S222A Q143R/S82A 1203.0 191.216% 3.05 2 Gla swap FIX/T128N/P129A/ Gla swap FIX/ 1703.2 145.2 9% 4.322 S222A/Q286R T[128]N/P[129]A/ S82A/Q143R Q286R/S222A/H257AQ143R/S82A/H117A 2592.0 806.5 31% 6.58 4 Q286R/M298Q Q143R/M156Q 299.362.9 21% 0.76 7 Q286R/M298Q† Q143R/M156Q† 287.3 26.6 9% 0.57 20Q286R/M298Q§ Q143R/M156Q§ 395.1 56.4 14% 0.79 3 Gla swap FIX/Q286R/M298QGla swap FIX/Q143R/M156Q 281.6 43.2 15% 0.72 3 Gla swap FIX/Q286R/M298Q†Gla swap FIX/Q143R/M156Q† 238.2 21.6 9% 0.47 3 T128N/P129A/Q286R/M298QT[128]N/P[129]A/Q143R/M156Q 283.7 49.4 17% 0.72 13T128N/P129A/Q286R/M298Q† T[128]N/P[129]A/Q143R/M156Q† 283.7 77.6 27%0.56 3 Gla swap FIX/ Gla swap FIX/ 508.2 197.0 39% 1.29 3T128N/P129A/Q286R/ T[128]N/P[129]A/Q143R/ M298Q M156Q Gla swap FIX/ Glaswap FIX/ 325.2 82.2 25% 0.65 2 T128N/P129A/Q286R/T[128]N/P[129]A/Q143R/M156Q† M298Q† {Gla swap FIX/E40L}/ {Gla swapFIX/E[40]L}/ 286.7 2.4 1% 0.73 2 Q286R/M298Q Q143R/M156Q {Gla swapFIX/K43I}/ {Gla swap FIX/K[43]I}/ 244.3 29.8 12% 0.62 5 Q286R/M298QQ143R/M156Q {Gla swap FIX/K43I}/ {Gla swap FIX/K[43]I}/ 219.3 13.7 6%0.44 2 Q286R/M298Q† Q143R/M156Q† {Gla swap FIX/Q44S}/ {Gla swapFIX/Q[44]S}/ 271.4 12.4 5% 0.69 2 Q286R/M298Q Q143R/M156Q {Gla swapFIX/M19K}/ {Gla swap FIX/M[19]K}/ 309.6 0.79 1 Q286R/M298Q Q143R/M156QGla swap FIX/S52A/S60A/ Gla swap FIX/ 253.6 0.50 1 Q286R/M298Q†S[52]A/S[60]A/Q143R/M156Q† {Gla swap {Gla swap FIX/M[19]K/ 339.3 100.830% 0.86 2 FIX/M19K/E40L/K43I/Q44S}/ E[40]L/K[43]I/Q[44]S}/ Q286R/M298QQ143R/M156Q {Gla swap {Gla swap FIX/K[43]I}/ 222.5 10.7 5% 0.44 2FIX/K43I}/T128N/P129A/ T[128]N/P[129]A/Q143R/M156Q† Q286R/M298Q†S222A/H257A/Q286R/ S82A/H117A/Q143R/M156Q 313.9 0.80 1 M298QT128N/P129A/S222A/ T[128]N/P[129]A/S82A/H117A/ 653.0 127.9 20% 1.66 4H257A/Q286R/M298Q Q143R/M156Q T128N/P129A/S222A/T[128]N/P[129]A/S82A/H117A/ 327.7 23.2 7% 0.65 2 H257A/Q286R/M298Q†Q143R/M156Q† S52A/S60A/S222A/H257A/Q286R/ S[52]A/S[60]A/S82A/H117A/447.6 117.6 26% 1.14 3 M298Q Q143R/M156Q Q286R/M298Q/Q366NQ143R/M156Q/Q217N 324.1 77.9 24% 0.82 3 T128N/P129A/Q286R/M298Q/T[129]N/P[129]A/Q143R/M156Q/ 345.8 24.2 7% 0.69 3 Q366N† Q217N† {Glaswap {Gla swap 404.4 48.0 12% 0.80 3 FIX/K43I}/Q286R/M298Q/Q366N†FIX/K[43]I}/Q143R/M156/Q217N† {Gla swap FIX/K43I}/ {Gla swapFIX/K[43]I}/ 319.1 71.8 22% 0.63 2 T[128]N/P[129]A/Q286R/M298Q/T[128]N/P[129]A/Q143R/M156Q/ Q366N† Q217N† Q286R/H373F Q143R/H224F 620.8133.4 3% 1.58 2 T128N/P129A/Q286R/H373F T[128]N/P[129]A/Q143R/H224F590.4 104.2 18% 1.50 4 Q286R/M298Q/H373F Q143R/M156Q/H224F 152.1 7.2 5%0.39 3 T128N/P129A/Q286R/M298Q/ T[128]N/P[129]A/Q143R/M156Q/ 182.6 43.224% 0.46 5 H373F H224F M298Q/H373F M156Q/H224F 81.7 10.5 13% 0.21 2T128N/P129A/M156Q/H224F† T[128]N/P[129]A/ 89.1 3.8 4% 0.18 2M156Q/H224F† V21D/Q143R/E154V/M156Q V21D/Q143R/E154V/M156Q 85.0 14.7 17%0.22 13 S222A/T239V S82A/T99V 967.3 282.6 29% 2.46 5 Gla swap FIX/ Glaswap FIX/ 2438.4 269.4 11% 6.19 2 S222A/T239V/Q286R S82A/T99V/Q143R Glaswap FIX/ Gla swap FIX/ 1343.5 507.1 38% 2.67 3 S222A/T239V/Q286R†S82A/T99V/Q143R† T239V/Q286R/M298Q T99V/Q143R/M156Q 3626.9 1465.9 40%9.21 4 Gla swap FIX/ Gla swap FIX/ 483.7 65.6 14% 1.23 2T239V/Q286R/M298Q T99V/Q143R/M156Q Gla swap FIX/ Gla swap FIX/ 314.30.62 1 T239V/Q286R/M298Q† T99V/Q143R/M156Q† T128N/P129A/T[128]N/P[129]A/ 266.4 52.1 20% 0.53 2 T239V/Q286R/M298Q†T99V/Q143R/M156Q† S222A/T239V/H257A/ S82A/T99V/H117A/Q143R/M156Q 469.3133.2 28% 1.19 6 Q286R/M298Q T128N/P129A/ T[128]N/P[129]A/ 326.5 55.317% 0.65 2 S222A/T239V/H257A/ S82A/T99V/H117A/Q143R/M156Q† Q286R/M298Q†T239V/Q286R/H373F T99V/Q143R/H224F 630.6 194.0 31% 1.60 3T128N/P129A/T239V/Q286R/ T[128N]/P129]A/T99V/Q143R/ 121.2 25.8 21% 0.244 M298Q/H373F† M156Q/H224F† V158D/T239I/E296V/M298QV21D/T99I/E154V/M156Q 179.5 50.5 28% 0.46 5 T239I/Q286R T99I/Q143R5823.0 2185.5 38% 14.79 9 S222A/T239I S82A/T99I 1149.8 12.8 1% 2.92 2GlaSwapFIX/S222A/T239I/Q286R Gla swap FIX/ 3313.1 130.3 4% 8.41 2S82A/T99I/Q143R T239I/Q286R/M298Q T99I/Q143R/M156Q 1611.4 185.9 12% 4.092 Gla swap FIX/ Gla swap FIX/ 1171.3 104.5 9% 2.97 2 T239I/Q286R/M298QT99I/Q143R/M156Q T128N/P129A T[128N]/P129]A 917.0 60.5 7% 1.82 3T239I/Q286R/M298Q† T99I/Q143R/M156Q† S222A/T239I/H257A/Q286R/S82A/T99I/H117A/Q143R/M156Q 1223.6 18.9 2% 3.11 2 M298QT239I/Q286R/H373F T99I/Q143R/H224F 1007.6 29.8 3% 2.56 2V158D/T239V/E296V/M298Q V21D/T99V/E154V/M156Q 67.7 16.6 24% 0.17 4V158D/T239V/E296V/M298Q† V21D/T99V/E154V/M156Q† 67.1 0.13 1 T239V/Q286RT99V/Q143R 1787.9 106.3 6% 4.54 2 T239I/Q286R/M298Q/H237FT99I/Q143R/M156Q/H224F 370.4 3.0 1% 0.94 2 T128N/P129AT239I/Q286R/M298QT[128]N/P[129]AT99I/Q143R/ 316.6 24.7 8% 0.63 2 H237F† M156Q/H224F†S222A/H257S/Q286R/M298Q S82A/H117S/Q143R/M156Q 526.7 1.34 1S222A/Q286R/M298Q/H373F S82A/Q143R/M156Q/H224F 163.2 46.9 29% 0.41 4 Glaswap FIX/S222A/ Gla swap FIX 163.5 58.2 36% 0.42 4 Q286R/M298Q/H373FS82A/Q143R/M156Q/H224F S222A/Q286R/M298Q S82A/Q143R/M156Q 308.4 119.939% 0.78 4 Gla swap FIX/ Gla swap FIX 266.3 104.2 39% 0.68 4S222A/Q286R/M298Q S82A/Q143R/M156Q T128N/P129A/A175S/Q366VT[128]N/P[129]A/A39S/Q217V 332.6 56.2 17% 0.84 3 A122N/G124S/A175S/Q366VA[122]N/G[124]S/A39S/Q217V 336.1 11.0 3% 0.85 3 T128N/P129A/A175S/S222AT[128]N/P[129]A/A39S/S82A 1913.4 4.86 1 A122N/G124S/A175S/S222AA[122]N/G[124]S/A39S/S82A 1548.6 394.1 25% 3.93 2T128N/P129A/A175S/Q286R T[128]N/P[129]A/A39S/Q143R 9545.5 2797.3 29%24.24 2 A122N/G124S/A175S/Q286R A[122]N/G[124]S/A39S/Q143R 6923.3 17.581 Gla swap FIX/ Gla swap FIX/ 587.3 5.1 1% 1.49 2 S222A/Q286R/H373FS82A/Q143R/H224F H257A/Q286R/M298Q H117A/Q143R/M156Q 390.8 0.99 1 Glaswap FIX/ Gla swap FIX/ 6486.4 148.2 2% 16.47 2 T128N/P129A/A175S/T[128]N/P[129]A/A39S/S82A/ S222A/Q286R Q143R Gla swap FIX/ Gla swap FIX/5524.0 1434.1 26% 14.03 2 A122N/G124S/A175S/ A[122]N/G[124]S/A39S/S82A/S222A/Q286R Q143R T128N/P129A/A175S/ T[128]N/P[129]A/A39S/Q143R/ 2311.8520.7 23% 5.87 2 Q286R/M298Q M156Q A122N/G124S/A175S/A[122]N/G[124]S/A39S/Q143R/ 1954.2 450.7 23% 4.96 2 Q286R/M298Q M156QT128N/P129A/A175S/ T[128]N/P[129]A/A39S/S82A/ 3212.9 1140.7 36% 8.16 2S222A/H257A/Q286R/M298Q H117A/Q143R/M156Q A122N/G124S/A175S/A[122]N/G[124]S/A39S/S82A/ 2972.8 751.2 25% 7.55 2S222A/H257A/Q286R/M298Q H117A/Q143R/M156Q T128N/P129A/A175S/T[128]N/P[129]A/A39S/Q143R/ 1132.4 441.3 39% 2.88 2 Q286R/M298Q/H373FM156Q/H224F A122N/G124S/A175S/ A[122]N/G[124]S/A39S/Q143R/ 1000.1 184.318% 2.54 2 Q286R/M298Q/H373F M156Q/H224F V158D/Q286R/E296V/M298Q/V21D/Q143R/E154V/M156Q/ 62.1 10.5 17% 0.16 11 H373F H224FM298Q/Q366N/H373F† M156Q/Q217N/H224F 90.8 4.8 5% 0.18 2T239V/M298Q/H373F† T99V/M156Q/H224F 46.6 7.9 17% 0.09 2T239I/M298Q/H373F† T99I/M156Q/H224F 178.7 29.7 17% 0.36 2T128N/P129A/Q286R/M298Q/ T[128]N/P[129]A/Q143R/M156Q 148.3 12.9 9% 0.292 Q366N/H373F† Q217N/H224F T239V/Q286R/M298Q/Q366N†Q143R/M156Q/Q217N/T99V 252.2 40.9 16% 0.50 2 T239I/Q286R/M298Q/Q366N†T99I/Q143R/M156Q/Q217N 813.2 105.1 13% 1.62 2 †produced in CHOX cells§produced in CHOX stable cell line clone 52-5F7

Example 6 In Vivo Assessment of FVIIa Polypeptide Procoagulant Activity

Mouse models of hemophilia A were established to assess the procoagulantactivity of FVIIa polypeptides. Hemophilia A was induced in CD-1 mice byintraperitoneal administration of anti-FVIII antibodies, followed bysurgical removal of the tips of the tails to initiate bleeding. Micedeficient in FVIII (FVIII^(−/−) mice) also were used, but were nottreated with anti-FVIII antibodies. The mice were then treated withFVIIa polypeptide and the amount of blood lost in 20 minutes wasmeasured to determine the procoagulant activity of the FVIIapolypeptides.

A. In Vivo Assessment of Wild-Type FVIIa Procoagulant Activity

A mouse model of hemophilia A was established to assess the procoagulantactivity of FVIIa polypeptides. Hemophilia A was induced in CD-1 mice byadministration of anti-FVIII antibodies, followed by surgical removal ofthe tips of the tails to initiate bleeding. The mice were then treatedwith FVIIa polypeptide and the time taken to stop bleeding, and theamount of blood lost during this time, was measured to determine theprocoagulant activity of the FVIIa polypeptides.

Male CD-1 mice were anesthetized by intraperitoneal administration ofboth thiobarbital sodium at 100 mg/kg, and ketamine at 100 mg/kg.Lidocaine was administered by subcutaneous injection into the ventralneck to reduce sensitivity. The trachea and carotid artery werecannulated through a small skin incision in the neck to facilitateunrestricted breathing and the administration of anti-Factor VIIIantibody, recombinant human Factor VIIa (rhFVIIa) and/or modified FVIIpolypeptides.

Cannulated mice were administered 3.76 mg sheep-anti-human-FVIIIantibody (Affinity Biologicals, lot IG129R4, 612 mouse BU/ml) in 40 μL.This dose was determined by conducting an initial dose responseexperiment with the antibody (using 0.376, 0.94, 1.88 and 3.76 mg ofanti-human-FVIII), and assessing blood loss and bleeding time. After 20minutes, the tails of the mice were placed in 15 mL tubes containing 39°C. phosphate buffered saline (PBS) for a period of 10 minutes. At 30minutes, the tails were briefly removed from the PBS solution and thelast 5 mm of the tails were severed to initiate bleeding. The time atwhich bleeding began was noted. The tails were then returned to the tubecontaining 39° C. PBS and allowed to bleed for 5 minutes (pre-bleed) toensure that the mice had responded to the anti-FVIII antibody. Followingthe pre-bleed, the mice were administered FVIIa polypeptides or thevehicle in which the FVIIa proteins were prepared and delivered. FVIIapolypeptides were diluted in either PBS or a buffer composed of 52 mMsodium chloride, 10.2 mM calcium chloride dehydrate, 9.84 mMglycylglycine, 0.01% polysorbate 80 and 165 mM mannitol. The FVIIapreparations were administered at either 1, 3 or 10 mg/kg, in a volumeequivalent to 3 mL/kg, via the carotid cannulae and the tails wereplaced in fresh tubes containing 39° C. PBS. The bleeding was monitoredfor a period of 20 minutes and the times at which bleeding stopped werenoted. The total bleeding time was calculated as the sum of the durationof bleeding during the pre-bleed, and the duration of bleeding followingadministration of FVIIa polypeptides, or PBS or buffer.

To determine the amount of blood lost during the bleeding episodes, thecontents of the 15 mL tubes were assayed for hemoglobin content. TritonX-100 was diluted 1 in 4 in sterile water and 100 μL was added to 1 mLof the samples to cause hemolysis. The absorbance of the samples wasthen measured at a wavelength of 546 nm. To calculate the amount ofblood lost, the absorbance was read against a standard curve generatedby measuring the absorbance at 546 nm of known volumes of murine blood,diluted in PBS and hemolysed as above with Triton X 100.

An experiment was conducted comparing rhFVIIa generated as describedabove with the commercially available recombinant human FVIIa(NovoSeven®, Novo Nordisk) and blood loss was assessed followingadministration of a 3 mg/kg dose of each protein. The blood loss in thevehicle group (buffer, n=15) was 671.9±57.89 μl over the 20 minuteperiod. This was reduced by the rhFVIIa produced by Catalyst Biosciencesto 264.1±56.59 μl and by NovoSeven® to 273.7±53.93 μl (n=14). Thisexperiment demonstrated equivalency between the two proteins.

B. Analysis of the Coagulant Activity of FVIIa Variants in CD-1 Micewith Induced Hemophilia A

Initial experiments were carried out to determine the dose required andtime and duration of effect of anti-human-FVIII antibodies when given bythe intraperitoneal route to induce hemophilia in CD-1 mice. For thefirst lot of anti-FVIII (lot 1; Affinity Biologicals, lot IG129R4), thiswas based initially on the dose used for the cannulation experiments,described above. The dose determined to cause a hemophilic state(uncontrolled bleeding over a 20 minute assay period) was 7.54 mg/mouse(80 μl of a 94.25 mg/ml stock solution). This lot had a neutralizingactivity of 612 mouse BU/ml. For the second lot of anti-human FVIII (lot2; Affinity Biologicals, lot IG1577R2, neutralizing activity of 474mouse BU/ml) the dose used was 11.98 mg/mouse (120 μl of a 99.8 mg/mlstock solution) and was administered at 6 hours prior to tail cut.

To induce hemophilia, male CD-1 mice (25-35 g) were dosedintraperitoneally with lot 1 or lot 2 of anti-FVIII prior to theexperiment. Male CD-1 and FVIII^(−/−) mice were anesthetized byintraperitoneal administration of a ketamine/xylazine cocktail (45 mg/mLand 3.6 mg/mL, respectively, in saline) and placed on a heated platform(39° C.) to ensure there was no drop in body temperature. The procedureroom was kept at a temperature of 82° F. Ten minutes prior to tail cutthe tail was immersed in 10 mls of pre-warmed PBS (15 ml centrifugetube; 39° C.). Eight to ten mice were injected with recombinant humanFVIIa (Novoseven®, Novo Nordisk) or modified FVII polypeptides dilutedin a buffer composed of 52 mM sodium chloride, 10.2 mM calcium chloridedehydrate, 9.84 mM glycylglycine, 0.01% polysorbate 80 and 165 mMmannitol via the tail vein in a single injection. Vehicle only also wasinjected into a group of mice as a control. If the injection was missed,the animal was excluded from the study. Injection with FVIIa polypeptideor vehicle was made 5 minutes prior to tail cut. A tail cut was madeusing a razor blade 5 mm from the end of the tail and blood wascollected into PBS for a period of 20 minutes. At the end of thecollection period, total blood loss was assessed. The collection tubeswere mixed and a 1 ml aliquot of each sample was taken and assayed forhemoglobin content. Triton X-100 was diluted 1 in 4 in sterile water and100 μL was added to the 1 mL samples to cause hemolysis. The absorbanceof the samples was then measured at a wavelength of 546 nm. To calculatethe amount of blood lost, the absorbance was read against a standardcurve generated by measuring the absorbance at 546 nm of known volumesof murine blood, diluted in PBS and hemolysed as above with Triton X100.

1. Dose Response Study Assessing Wild-Type FVIIa Coagulant Activity

A dose response study in which 0.3, 1 or 3 mg/kg of wild-type FVIIa wasassessed also was performed. Mice that received the vehicle lost1002.3±60.71 μL in the 20 minute assay. This was reduced significantlyin mice that were administered 3 mg/kg of wild-type FVIIa, to415.5±90.85 μL (p<0.05 using Kruskal-Wallis followed by Dunn's posttest). Reducing the dose to 1 mg/kg resulted in blood loss of679.57±83.95 μL and a lower dose of 0.3 mg/kg resulted in blood loss of852.42±94.46 μL.

2. Initial Analysis of FVIIa Variant Coagulant Activity

The vehicle only injection was used as a control. Mice that received thevehicle only lost 915.2±105.6 μL in the 20 minute assay, which wasreduced to 352±99.86 μL (mean±S.E.M) if mice were administeredrecombinant human FVIIa. The amount of blood lost was reduced evenfurther to 165.8±48.41 μL if the mice were administered Q286R-FVIIa(i.e. FVIIa containing the Q286R mutation), to 141.3±43.77 μL withQ286R/M298Q-FVIIa or to 129.5±36.64 μL with V158D/E296V/M298Q-FVIIa.Mice administered S222A-FVIIa also exhibited reduced blood loss (to225.7±62.75 μL) compared to mice administered wild-type FVIIa.Administration of Gla Swap FIX-FVIIa, Q366V-FVIIa or A122N/G124S-FVIIaresulted in approximately the same amount of blood loss as that observedin mice given recombinant human FVIIa (334.6±54.95 μL, 321.7±102.6 μLand 329.8±83.91 μL respectively), while mice that were administeredH257A-FVIIa, S222A/Q286R-FVIIa, or H257A-FVIIa appeared to have slightlygreater blood loss (390±107 μL, 447.3±127.7 μL and 443.7±139.5 μLrespectively).

3. Dose Response Assessing FVIIa Variant Coagulant Activity

A dose response study in which 0.1, 0.3, 1 or 3 mg/kg of Q286R-FVIIa,S222A-FVIIa, Q286R/M298Q-FVIIa or V158D/E296V/M298Q-FVIIa wereadministered to the mice. Mice that received the vehicle only lost915.2±105.6 μL of blood in the 20 minute assay. This was reducedsignificantly in mice that were administered 3 mg/kg of any ofQ286R-FVIIa (141.3±43.77 μL), S222A-FVIIa (225.7±62.75 μL) orQ286R/M298Q-FVIIa (129.5±36.64 μL) (p<0.05 using Kruskal-Wallis followedby Dunn's post test). Reducing to the dose to 1 mg/kg resulted in bloodloss of 641±96.48 μL in mice that received Q286R-FVIIa and 487.92±92.07μL in mice that received S222A-FVIIa. Lower doses of this FVIIa variantresulted in approximately the same blood loss (817.71±107.94 μL and900.34±115.77 μL for Q286R-FVIIa and S222A-FVIIa respectively) asobserved in mice that received the vehicle control. In contrast, micethat received 1 mg/kg Q286R/M298Q-FVIIa had significantly reduced bloodloss (69.36±15.55 μL) compared to the vehicle only control mice. Atlower doses of 0.3 mg/kg and 0.1 mg/kg, blood loss was 538.3±94.04 μLand 664±121.6 μL, respectively. Mice receiving 0.3, 1 and 3 mg/kg ofV158D/E296V/M298Q-FVIIa had blood loss of 754.49±121.6 μL, 481.95±114.22μL and 133.25±50.09 μL respectively.

Additional dose response studies were carried out for Q286R-FVIIa,Q286R/M298Q-FVIIa and V158D/E296V/M298Q-FVIIa and the data combined withthose above. In the experiments assessing the effect of Q286R-FVIIa, thegroup that received the vehicle lost 833.61±73.95 μL of blood. This wassignificantly reduced to 196.71±49.18 μL in mice that were treated with3 mg/kg of Q286R-FVIIa. Reducing the dose to 1 mg/kg resulted in bloodloss of 577.78±66.29 μL. When mice were dosed with 0.1 and 0.3 mg/kg ofQ286R-FVIIa the blood loss was produced was similar to the vehiclecontrol value (739.58±104.28 μL and 806.63±65.17 μL, respectively). Inthe experiments assessing the effect of Q286R/M298Q-FVIIa, the vehiclegroup produced blood loss of 902.42±88.04 μL, which was significantlyreduced by treatment with both 1 and 3 mg/kg Q286R/M298Q-FVIIa to145.17±38.89 μL and 140.76±33.36 μL of blood loss. Reducing the dose to0.1 and 0.3 mg/kg resulted in blood loss values of 664.03±121.62 μL and551.94±67.60 μL respectively. In the experiments assessingV158D/E296V/M298Q-FVIIa the vehicle control group demonstrated bloodloss of 966.64±57.97 μL, which was significantly reduced by treatmentwith V158D/E296V/M298Q-FVIIa at 3 mg/kg to 128.19±27.73 μL. Reducing thedose to 1 mg/kg led to blood loss of 565.50±65.78 μL and reducing thedose further to 0.3 and 0.1 mg/kg produced blood loss which was similarto the control group (811.16±71.87 μL and 893.62±106.73 μL). Statisticalanalysis was made by Kruskal Wallis followed by Dunn's post test andsignificance was accepted when p<0.05.

4. Coagulant Activity of H216A-FVIIa, H373F-FVIIa, Q366D-FVIIa andQ366N-FVIIa at a Dose of 3 mg/kg.

A set of experiments tested H216A-FVIIa, H373F-FVIIa, Q366D-FVIIa andQ366N-FVIIa at a dose of 3 mg/kg. The vehicle only injection was used asa control. Mice that received the vehicle lost 915.2±105.6 μL in the 20minute assay. This was reduced even further to 211.1±67.70 μL if micewere treated with H373F-FVIIa. Blood loss was less affected upontreatment with H216A-FVIIa, Q366D-FVIIa and Q366N-FVIIa, with values of558.6±66.22, 577.1±151.4 and 477.1±112.6 μL respectively.

4. Coagulant Activity of Q286R/M298Q/Gla Swap FIX-FVIIa,S222A/H257A/Q286R/M158Q-FVIIa, Q286R/M298Q/K341D-FVIIa andQ286R/M298Q/H373F-FVIIa at a Dose of 3 mg/kg.

The coagulant activity of Q286R/M298Q/Gla swap FIX-FVIIa,S222A/H257A/Q286R/M158Q-FVIIa, Q286R/M298Q/K341D-FVIIa andQ286R/M298Q/H373F-FVIIa at a dose of 3 mg/kg was assessed in the CD-1hemophilia mouse model. The vehicle only injection was used as acontrol. Mice that received the vehicle lost 803±92.18 μL in the 20minute assay. This was reduced by treatment withS222A/H257A/Q286R/M158Q-FVIIa to 118.6±63.27 μL. Treatment withQ286R/M298Q/Gla swap FIX-FVIIa at 3 mg/kg reduced the blood losscompared to the vehicle group from 888.89±104.76 μL to 171.83±62.061 μL.In the experiments assessing Q286R/M298Q/K341D-FVIIa andQ286R/M298Q/H373F-FVIIa the blood loss from the vehicle group was813.1±82.66 μL. This was reduced to 39.42±5.53 μL blood loss followingtreatment with Q286R/M298Q/H373F-FVIIa. Q286R/M298Q/K341D-FVIIa appearedto be less effective in the assay, resulting in blood loss of636.7±121.6 μL.

5. Dose Response Study to Assess Coagulant Activity ofS222A/H257A/Q286R/M158Q-FVIIa and Q286R/M298Q/H373F-FVIIa

The dose response to S222A/H257A/Q286R/M158Q-FVIIa at 0.3, 0.5, 1 and 3mg/kg was assessed. Mice that received the vehicle lost 832.48±71.70 μLin the 20 minute assay. This was reduced significantly in mice that wereadministered 3 mg/kg of S222A/H257A/Q286R/M158Q-FVIIa, to 118.63±63.27μL (p<0.05 using Kruskal-Wallis followed by Dunn's post test). Reducingthe dose to 1 and 0.5 mg/kg resulted in a significant reduction in bloodloss (202.69±77.60 μL and 366.52±106.21 μL) and a lower dose of 0.3mg/kg resulted in blood loss comparing more to vehicle levels(742.04±112 μL). A dose response to Q286R/M298Q/H373F-FVIIa at 0.1, 0.3,1 and 3 mg/kg was also assessed and in this experiment mice thatreceived the vehicle had blood loss of 813.15±82.66 μL. This was reducedsignificantly in mice that were treated with 3 mg/kg ofQ286R/M298Q/H373F-FVIIa, to 39.42±5.52 μL (p<0.05 using Kruskal-Wallisfollowed by Dunn's post test). Reducing the dose to 1 and 0.3 mg/kg ledto blood loss values of 208.10±105.12 and 508.9±155.8 μL respectively.The lowest dose tested of 0.1 mg/kg produced blood loss that wasapproaching vehicle control levels (733.5±152.88 μL).

C. Analysis of FVIIa Coagulant Activity in FVIII^(−/−) Mice

A mouse model of hemophilia A using mice deficient in FVIII (FVIII^(−/−)mice) also was used to assess the coagulant activity of FVIIapolypeptides, using the same protocols as described above except thatthe mice were not treated with anti-FVIII antibodies.

1. Dose Response Study Assessing Wild-Type FVIIa Coagulant Activity

Dose response studies to assess the coagulant activity of NovoSeven® andwild-type rhFVIIa in FVIII^(−/−) mice at 0.3, 1, 3 and 6 mg/kg wereperformed. In the NovoSeven® experiment, the blood loss in the vehiclegroup was 912.79±38.32 μL, which was significantly reduced by NovoSeven®treatment at 6 and 3 mg/kg (to 361.74±55.28 μL and 586.98±60.56 μL;p<0.05 using Kruskal-Wallis followed by Dunn's post test). Reducing thedose to 1 mg/kg resulted in blood loss of 674.84±46.88 μL and at thelowest dose tested the value was 801.08±41.39 μL. In the wild-typerhFVIIa experiment, the vehicle control group produced blood loss of904.08±15.38 μL. This was reduced significantly (p<0.05 usingKruskal-Wallis followed by Dunn's post test) by wild-type rhFVIIa at 6mg/kg to 451.04±74.17 μL. Reducing the dose to 3 mg/kg produced a bloodloss value of 695.75±60.50 μL while lowering the dose further to 1 and0.3 mg/kg resulted in blood loss values near and at vehicle controllevels (846.08±34.17 μL and 936.43±31.39 μL, respectively).

2. Dose Response Assessing Q143R-FVIIa, S222A-FVIIa, Q286R/M298Q-FVIIaand V158D/E296V/M298Q-FVIIa Coagulant Activity

The first set of experiments tested recombinant human FVIIa (NovoSeven®,Novo Nordisk), V158D/E296V/M298Q-FVIIa, Q286R-FVIIa and S222A-FVIIa at 3mg/kg. The vehicle only injection was used as a control. Mice receivingvehicle only had blood loss of 942.9±27.37 μL in the 20 minute assay.Treatment with NovoSeven® FVII, V158D/E296V/M298Q-FVIIa, Q143R-FVIIa andS222A-FVIIa reduced blood loss to 468±55.9 μL, 302.38±73.12 μL,697.26±92.22 μL and 675.07±35.29 μL respectively. Q143R-FVIIa, whentested again in FVIII^(−/−) mice at 3 mg/kg, demonstrated a reducedblood loss of 754.84±60.96 μL compared to the vehicle control group(935.54±51.96 μL). When assessed at 5 mg/kg, Q143R-FVIIa produced afurther reduction in blood loss to 445.87±79.62 μL compared to thevehicle control group (960.42±24.5 μL).

The second sets of experiments were dose response studies in whichV158D/E296V/M298Q-FVIIa and Q286R/M298Q-FVIIa were assessed at 0.3, 1and 3 mg/kg. Treatment with V158D/E296V/M298Q-FVIIa at 3 mg/kg resultedin significant reduction in blood loss (375.62±74.22 μL) compared to thevehicle control (960.42±24.5 μL; p<0.05 using Kruskal-Wallis followed byDunn's post test). Reducing the dose to 1 and 0.3 mg/kg led to bloodloss values nearer control levels, of 834.76±54.38 μL and 841.62±68.99μL respectively. A second experiment which assessed a different lot ofV158D/E296V/M298Q-FVIIa produced a vehicle control blood loss value of912.79±38.32 μL, which was significantly inhibited at 3 and 1 mg/kg(247.24±35.17 μL and 628.30±37.36 μL; p<0.05 using Kruskal-Wallisfollowed by Dunn's post test). Treatment with a lower dose ofV158D/E296V/M298Q-FVIIa produced blood loss that was approaching controlvalues (841.85±19.32 μL). In the experiment assessing the effect ofQ286R/M298Q-FVIIa in FVIII^(−/−), mice the vehicle group produced bloodloss of 941.39±35.18 μL. This was significantly inhibited at a dose of 3mg/kg, resulting in blood loss of 258.92±59.82 μL. At lower doses of 1and 0.3 mg/kg the levels of blood loss produced were 616.82±78.43 μL and924.9±38.01 μL, respectively.

D. Analysis of the Coagulant Activity of Additional FVIIa Variants inUsing the Induced Hemophilia Model

The coagulant activity of several FVIIa variants was assessed using theInduced Hemophilia Model (IHM) with CD-1 mice described in Example 6.B,above. The protocol was the same as described above, except for theassessment of the T128N/P129A-FVIIa variant and the M156Q/H224F-FVIIavariant, in which a different lots (third and fourth, respectively) ofhuman anti-FVIII were used. For the third lot of anti-human FVIII (lot3; Affinity Biologicals, lot IG1603R1, neutralizing activity of 418mouse BU/ml) the dose used was 12.17 mg/mouse (120 μl of a 101.4 mg/mlstock solution), which was administered at 18 hours prior to tail cut.For the fourth lot of anti-human FVIII (lot 4; Affinity Biologicals, lotIG1639R1, neutralizing activity of 875 mouse BU/ml) the dose used was8.04 mg/mouse (80 μl of a 100.45 mg/ml stock solution. The blood losswas measured as described above, and presented below in Table 19 as thepercent inhibition (calculated using the average values for the variantof interest and dividing by the vehicle group), and the ED50 value(determined using non linear regression analysis (using GraphPad Prism®software, GraphPad Software, Inc.), constraining the top and bottom ofthe response curve with the blood loss observed with vehicle-treated andnormal control animals, respectively.

TABLE 19 IHM Blood Loss; IHM Blood Mutation Mutation % Inhibition Loss;(mature FVII numbering) (Chymotrypsin numbering) (1.5 mg/kg) n ED50(mg/kg) n WT (NovoSeven ®) WT (NovoSeven ®) 61 1 0.7 1 WT WT 59 1 0.75 1T128N/P129A T[128]N/P[129]A 78 1 0.5 1 Gla swap FIX Gla swap FIX 63 1A122N/G124S A[122]N/G[124]S 64 1 V158D/E296V/M298Q V21D/E154V/M156Q 86 30.5 3 Q286R Q143R 76 2 0.55 2 S222A S82A 75 1 0.45 1 H257S H117S 50 1H373F H224F 78 1 Q366V Q217V 64 1 A175S A39S 58 1 K109N/A175SK[109]N/A39S 60 1 Q286R/H257A Q143R/H117A 58 1 Q286R/S222A Q143R/S82A 501 Q286R/S222A/H257A Q143R/S82A/H117A 68 1 Q286R/M298Q Q143R/M156Q 85 20.15 2 Q286R/M298Q† Q143R/M156Q† 89 1 0.16 2 Gla swap FIX/ Gla swap FIX/83 2 0.13 1 Q286R/M298Q Q143R/M156Q S222A/H257A/Q286R/S82A/H117A/Q143R/M156Q 85 1 0.2 1 M298Q H257S/Q286R/Q366VH117S/Q143R/Q217V 46 1 S222A/H257A/Q286R/ S82A/H117A/Q143R/Q217V 49 1Q366V Q286R/M298Q/Q366N Q143R/M156Q/Q217N 88 1 Q286R/H373F Q143R/H224F75 1 Q286R/M298Q/H373F Q143R/M156Q/H224F 95 1 0.15 1 M298Q/H373FM156Q/H224F 95 1 0.21 1 Glaswap Glaswap FIX/S82A/Q143R 85 1 0.4 1FIX/S222A/Q286R V158D/Q286R/E296V/ V21D/Q143R/E154V/M156Q 95 1 M298QT128N/P129A/Q286R/ T[128]N/P[129]A/Q143R/H224F/ 70 1 H373F †FVIIapolypeptide produced from CHOX cellsE. Analysis of the Coagulant Activity of Additional FVIIa Variants inUsing the Induced Hemophilia Model

The coagulant activity of several FVIIa variants was assessed using theInduced Hemophilia Model (IHM) with CD-1 mice described in Example 6.B,above. The protocol was the same as described above, except that forthese experiments lots 4 through 6 of anti-FVIII were used. The detailsfor these lots were as follows: lot 4 (detailed above), for the fifthlot (lot 5; Affinity Biologicals, lot IG1593R2, neutralizing activity of255 mouse BU/ml) the dose used was 12.25 mg/mouse (120 μl of a 102.1mg/ml stock solution), which was administered at 6 hours prior to tailcut. For the sixth lot (lot 6; Affinity Biologicals, lot IG1703R2,neutralizing activity of 685 mouse BU/ml) the dose used was 8.02mg/mouse (80 μl of a 100.2 mg/ml stock solution), which was administeredat 4 pm on the day prior to experiment. The blood loss was measured asdescribed above, and presented below in Table 20 as the percentinhibition (calculated using the average values for the variant ofinterest and dividing by the vehicle group), in each case all doses areshown with the corresponding inhibition values below, and the ED50 value(determined using non linear regression analysis (using GraphPad Prism®software, GraphPad Software, Inc.), constraining the top and bottom ofthe response curve with the blood loss observed with vehicle-treated andnormal control animals, respectively. The ‘n/group’ refers to the amountof mice per group whereas the ‘n’ in reference to the ED50 calculationrefers to the amount of experiments performed with the variant.

TABLE 20 IHM Blood Loss; % Inhibition at IHM each dose Blood (mg/kg)Loss; Mutation Mutation Dose (mg/kg) n/ ED50 (mature FVII numbering)(Chymotrypsin numbering) 0.1 0.3 1 group (mg/kg) n H257A/Q286RH117A/Q143R 53 7-10 M298Q M156Q 37 81 87 7-10 0.11 1 M298Q/T128N/P129AM156Q/T[128]N/P[129]A 39 79 93 7-9 0.1 1 Q286R/T128N/P129AQ143R/T[128]N/P[129]A 32 42 58 6-8 0.27 1 Gla Swap FIX/T128N/P129A/GlaswapFIX/T[128]N/P[129]A/ 27 45 62 6-8 0.27 S222A/Q286R S82A/Q143RQ286R/M298Q Q143R/M156Q 33 57 89 9-10 0.15 1 Q286R/M298Q Q143R/M156Q 3557 94 8-10 0.13 1 Q286R/M298Q Q143R/M156Q 32 62 95 8-9 0.14 1Q286R/M298Q Q143R/M156Q 44 65 95 9 0.09 1 Gla Swap FIX/Q286R/M298Q GlaSwap FIX/Q143R/M156Q 35, 57 80 7-10 0.14 1 T128N/P129A/Q286R/M298QT[128]N/P[129]A/ 47, 83 91 9-14 0.1 1 Q143R/M156Q Gla SwapFIX/T128N/P129A/ Gla Swap FIX/T[128]N/P[129]A/ 15 71 96 7-9 0.17 1Q286R/M298Q Q143R/M156Q {GlaswapFIX/K43I}/Q286R/{GlaswapFIX/K[43]I}/Q143R/ 49 65 80 7-9 0.08 1 M298Q M156QGlaswapFIX/S52A/S60A/ GlaswapFIX/S[52]A/S[60]A/ 38 71 88† 9-10 0.11 1Q286R/M298Q/ Q143R/M156Q/ {GlaswapFIX/K43I}/T128N/P129A/{GlaswapFIX/K[43]I}/T[128]N/ 23 58 84 9 0.17 1 Q286R/M298QP[129]A/Q143R/M156Q S222A/H257A/Q286R/M298Q/ S82A/H117A/Q143R/M156Q/ 2471 96 7-8 0.15 1 T128N/P129A T[128]N/P[129]A Q286R/M298Q/Q366NQ143R/M156Q/Q217N 41 79 96 7-8 0.1 1 Vehicle only: n = 17 T128N/P129A/T[128]N/P[129]A/ 40 53 84 6-8 0.09 1 Q286R/M298Q/Q366N Q143R/M156Q/Q217N{GlaswapFIX/K43I}/T128N/P129A/ {GlaswapFIX/K[43]I}/T[128]N/ 39 81 948-10 0.1 1 Q286R/M298Q/Q366N/ P[129]A/Q143R/M156Q/ Q217NT128N/P129A/Q286R/H373F T[128]N/P[129]A/Q143R/H224F 26 7-9T128N/P129A/Q286R/M298Q/ T[128]N/P[129]A/ 43 42 87 6-13 0.12 1 H373FQ143R/M156Q/H224F M298Q/H373F M156Q/H224F 27 46 82 7-8 0.21 1T128N/P129A/M298Q/H373F T[128]N/P[129]A/M156Q/ 48 77 85† 8-10 0.09 1H224F V158D/E296V/M298Q/Q286R V21D/E154V/M156Q/Q143R 27, 61 95 7-8 0.161 GlaswapFIX/S222A/Q286R/ GlaswapFIX/S82A/Q143R/T99V 30, 32 60 8-9 0.361 T239V Q286R/M298Q/T239V Q143R/M156Q/T99V 21 67 92 7 0.16 1GlaswapFIX/Q286R/M298Q/ GlaswapFIX/Q143R/M156Q/ 16 69 90 7-9 0.17 1T239V T99V T128N/P129A/T239V/ T[128]N/P[129]A/T99V/ 29 63 91 9 0.15 1Q286R/M298Q Q143R/M156Q/ S222A/T239V/H257A/ S82A/T99V/H117A/ 38 63 947-8 0.12 1 Q286R/M298Q Q143R/M156Q/ T128N/P129A/S222A/T239V/T[128]N/P[129]A/S82A/T99V/ 44 75 79 7-9 0.09 1 H257A/Q286R/M298QH117A/Q143R/M156Q T128N/P129A/T239V/ T[128]N/P[129]A/T99V/ 51 75 88 8-100.08 1 Q286R/M298Q/H373F Q143R/M156Q/H224F V158D/T239I/E296V/M298QV21D/T99I/E154V/M156Q 45 48 85 7-9 0.11 1 T128N/P129A/T[128]N/P[129]A/T99I/ 20 61 82 8-10 0.18 1 T239I/Q286R/M298Q Q143R/M156QV158D/T239V/E296V/M298Q V21D/T99V/E154V/M156Q 23 52 86 8-9 0.19 1T128N/P129A/T239I/ T[128]N/P[129]A/T99I/ 36 67 73 8-10 0.11 1Q286R/M298Q/H373F/ Q143R/M156Q/H224F M298Q/Q366N/H373F M156Q/Q217N/H224F33 80 88 8-10 0.11 1 T239V/M298Q/H373F T99V/M156Q/H224F 50 68 84 7-90.07 1 Vehicle only: n = 16 T239I/M298Q/H373F T99I/M156Q/H224F 56 74 969-10 0.07 1 T128N/P129A/Q286R/ T[128]N/P[129]A/Q143R/M156Q/ 51 87 92 7-90.1 1 M298Q/Q366N/H373F Q217N/H224F T239V/Q286R/M298Q/Q366NT99V/Q143R/M156Q/Q217N 45 62 80 7-9 0.08 1 T239I/Q286R/M298Q/Q366NT99I/Q143R/M156Q/Q217N  8 34 79 8-9 0.34 1 †Dose was 0.6 mg/kg

Example 7 Michaelis Menten Kinetics Constant Determination of theAmidolytic Activity of FVIIa on a Small Molecule Substrate

The amidolytic activity of the FVII variants can be assessed bymeasuring the Michaelis Menten kinetics constant of the FVIIapolypeptide on the peptidyl substrate Spectrozyme FVIIa(CH₃SO₂-D-CHA-But-Arg-pNA.AcOH). Such an assay can be performed asfollows.

Lipidated human purified tissue factor (Innovin, Dade Behring, VWRCat#68100-390) is included in the assay to provide for optimal activityof FVIIa. The TF-FVIIa complex cleaves Spectrozyme FVIIa as a highlyspecific chromogenic substrate releasing a paranitroaniline-chromophore(pNA), which can be monitored by measuring absorption at 405 nm. Enzymeactivity is determined by monitoring the absorbance at 405 nm of thefree pNA generated as a function of time.

The reactions are performed at three different enzyme concentrations.For the reaction, the FVIIa variants are first diluted to 40 nM in 1×direct assay buffer (100 mM Tris pH 8.4, 100 mM NaCl, 5 mM CaCl₂, 0.01%BSA) in a 1.7 mL tube (low adhesion microfuge tubes from ISCBioexpress). FVIIa is further diluted in the presence of TF (Innovin,Dade Behring) by diluting to 2 nM in a 12-well polypropylene reservoir(Axygen) as follows: 720 μl 5× direct buffer (500 mM Tris pH 8.4, 500 mMNaCl, 25 mM CaCl₂, 0.05% BSA), 180 μl 40 nM FVIIa, and 2700 μl 2×TF (6nM stock solution reconstituted in 10 mL water). The diluted protease isincubated for 5 minutes at room temperature. The 2 nM stock of FVIIa isfurther diluted in 2-fold serial dilutions to give a 1 nM and 0.5 nMstock of protease, respectively, also in the presence of TF. The serialdilution reactions are as follows: first, 1800 μl of 2 nM stock ofFVIIa/TF from above diluted into 360 μl 5× direct buffer, 900 μl 2×TF,and 540 μl water. This diluted stock is diluted again 1:1 into 1800 μl1×TF in direct buffer.

A dilution plate of the substrate Spectrozyme FVIIa (AmericanDiagnostica) is made. The stock solution of Spectrozyme FVIIa is made byreconstitution of the 50 moles vial in distilled water to 10 mM andstored at 4° C. Eighty μl (10 mM Spectrozyme FVIIa) and 60 μl+20 μlwater (7.5 mM Spectrozyme FVIIa) of the 10 mM Spectrozyme FVIIa areadded to wells in two adjacent columns of a 96-well polypropylene assayplate (Costar). The two wells are serially diluted 2-fold down each ofthe 8 wells of the respective column to make a series of 10× substrateconcentrations ranging from 10 mM to 78 μM substrate down the wells ofthe first column and from 7.5 mM to 58.6 μM substrate down the wells ofthe second column.

Five μl of each Spectrozyme FVIIa substrate dilution is added to a96-well clear half area assay plate (Costar). Forty five μl of each ofthe three FVIIa/TF dilutions are added to three groups of columns of thesubstrate series dilutions. During this step, care is taken to avoidintroducing bubbles into the wells of the assay. If bubbles areintroduced, they can be removed by pricking with a clean needle beforethe beginning of each assay. The plates are then mixed by shaking. Priorto initiation of the assay, the pathlength of the assay wells ismeasured using a Spectramax Gemini M5 plate reader spectrophotometer(Molecular Devices) by taking an endpoint reading and using thePathcheck feature of the SoftMax Pro software (Molecular Devices). Theincrease in absorbance at 405 nm is measured every 30 seconds for onehour at 37° C.

The SoftMax Pro software is used to convert the absorbance rate(milliunits/sec) to concentration of pNA released (μM/sec) by using thepathlength and the extinction coefficient of the pNA leaving group at405 nm, 9600 M⁻¹cm⁻¹. The conversion equation is as follows: Rate×(1/60×1000)×( 1/9600×Pathlength)×100000. The results for eachconcentration of protease are graphed using Graph Pad Prism softwarewith the substrate concentration on the X-axis and the determined μM/secrates on the Y-axis. Using Graph Pad Prism 4 software Km and Ymax isdetermined by fitting the data to a Michaelis Menten equation asfollows:Y=((k _(cat) K _(m)/1000000)×X×[E])/(1+(X+K _(m))

where;

-   -   X is the substrate concentration (μM)    -   Y is the enzyme activity (μM/sec)    -   k_(cat)K_(m) is the specificity constant (M⁻¹sec⁻¹)    -   K_(m) is the Michaelis constant (μM)    -   E is the enzyme concentration (μM)        Initial values of E=1, Km=X at 0.5×Y max and k_(cat)K_(m)=1000        were set.

Example 8 Assessment of the Potency of the Interaction Between FVIIaVariants and TFPI

The potency of the interaction between FVIIa polypeptides, such as thoseprovided herein, and TFPI, can be assessed using one or more assays. Inone example, the potency of the interaction between TFPI and a FVIIa/TFcomplex is assessed by measuring the level of inhibition of variousconcentrations of TFPI on the catalytic activity of a FVIIa/TF towards asubstrate, Spectrazyme VIIa. In another example, a high-throughputsurface plasmon resonance (SPR) assay can be used.

A. Determination of the IC₅₀ for TFPI Inhibition of FVIIa/TF

The potency of the interaction between TFPI and the FVIIa/TF complex wasassessed by measuring the level of inhibition of various concentrationsof TFPI on the catalytic activity of a FVIIa/TF towards a substrate,Spectrazyme VIIa. The concentration of TFPI that was required for 50%inhibition (IC₅₀) was calculated for each FVII variant, and a FVIIastandard.

A 96 well clear half area assay plate (Nunc) was pretreated by adding150 μl/well of 1× plate buffer (100 mM Tris pH 8.4, 100 mM NaCl, 0.01%BSA, 0.01% Tween-20) to each well and incubating the plate at 37° C. fora minimum of 1 hour. The buffer was removed completely by shaking andblotting the plate and centrifuging the plate upside down to remove theremaining buffer. The plate was air-dried for 1 hour, and stored at roomtemperature (RT).

In a 1.7 ml microfuge tube (low adhesion microfuge tube from ISCBioexpress), a mixture of FVIIa/TF was prepared in a total volume of 450μl by mixing 9 μl of 250 nM FVIIa (American Diagnostica, wild-type FVIIaor a respective variant to be tested) was mixed with 337.5 μl of 2×TF(Innovin; Dade Behring; lyophilized product resuspended in 10 mLdistilled water to generate 2×TF, which approximately equals 7 nM oflipidated TF), 90 μl 5× assay buffer (500 mM Tris pH 8.4, 500 mM NaCl,25 mM CaCl₂, 0.05% BSA) and 13.5 μl of water, resulting in a solutioncontaining 5 mM FVIIa and 5.2 nM TF. The mixture was incubated at roomtemperature for 5 minutes to allow the components to complex. To eachwell of 2 columns in the pretreated 96 well clear half area assay plate,25 μl of the respective FVIIa/sTF mixture was added and the plate wascovered to prevent evaporation.

Human Recombinant TFPI (R&D Systems) was initially dissolved in 33 μl50% glycerol (v/v) to make a 10 μM stock for storage at −20° C. The TFPIstock was further diluted to 1.5 μM in a final 1× buffer (100 mM Tris pH8.4, 100 mM NaCl, 5 mM CaCl₂, 0.01% BSA) in a polypropylene storageplate as follows: for each protease tested, 87.5 μl of a 1.5 μM solutionof TFPI was made by mixing 13.1 μl 10 μM TFPI with 17.5 μl 5× assaybuffer and 56.9 μl distilled water. Serial 3-fold dilutions of the TFPIsolution were made in 1× assay buffer by mixing 27.5 μl TFPI into 55 μl1× assay buffer, such that solutions containing 750 nM, 250 nM, 83.3 nM,27.8 nM, 9.26 mM, 3.1 nM, and 1.03 nM TFPI were generated. The finalwell of the series contained only 1× buffer as a control.

Twenty-five μl of each dilution of TFPI was added to 2 wells (i.e. induplicate) of 2 columns of the 96 well clear half area assay platecontaining the FVIIa/TF mixture, such that the protease mixture wasassayed in duplicate with each TFPI dilution. A solution of 1× assaybuffer without TFPI also was added to 2 wells containing the FVIIa/TFmixture as a negative control. The plate was agitated briefly and thencentrifuged at 3000 rpm for 5 minutes before incubation at 37° C. for1.5 hours.

A stock solution of Spectrazyme VIa (American Diagnostica) was preparedby reconstituting 50 μmoles in 5 ml distilled water to 10 mM and storingat 4° C. until use. Immediately prior to use, the solution was dilutedto 600 μM in distilled water. Following incubation of the assay platefrom above, 10 μl of the diluted Spectrazyme VIIa was added to each wellof the assay plate. The reactions were mixed and the plate was incubatedat 37° C. The increase in absorbance at 405 nm was measured every 30seconds for one hour at 37° C., and the absorbance rate were calculatedusing SoftMax Pro software (Molecular Devices).

To determine the degree of inhibition by TFPI, the absorbance rates ofprotease reactions containing TFPI were first divided by the absorbancerate of reactions containing no TFPI (the control sample) to obtain thefractional activity, and the log₁₀ of each TFPI concentration wasdetermined. Using GraphPad Prism Software, the log₁₀[TFPI] was plottedagainst the fractional activity for each protease, and a dose responsecurve was generated with a curve fit that assumed the top and bottom ofthe activity data are fixed at 1 and 0, respectively. The software wasused to determine TFPI inhibition as both the log IC₅₀ (pIC₅₀) value,and the absolute IC₅₀ (TFPI inhibition in nM) for each protease, and itsaverage and standard deviated was determined.

The level of inhibition of TFPI of each of the FVIIa variants in complexwith lipidated TF (Innovin; Dade Behring) was determined and expressedas the fold-increase of TFPI resistance compared to wild-type FVIIa(Table 21).

TABLE 21 Inhibition of FVIIa variants by TFPI TFPI Mutation (mature FVIIMutation (chymotrypsin fold numbering) numbering) resistance wt wt 1.0A292N/A294S A150N/A152S 2.4 A175S A39S 1.5 K109N K[109]N 0.7 A122N/G124SA[122]N/G[124]S 1.1 A122N/G124S/E394N/ A[122]N/G[124]S/E245N/P246A/ 1.0P395A/R396S R247SB. Surface Plasmon Resonance (SPR) Screening of FVIIa Variants forResistance to TFPI

The relative resistance of various FVIIa variants to inhibition by humanrecombinant soluble TFPI was evaluated using a high-throughput surfaceplasmon resonance (SPR) assay with the Biacore T100 instrument. Therelative resistance of FVIIa variants to inhibition by TFPI was assessedby measurement of the relative amount of FVIIa variant bound to solubleTFPI immobilized on a Biacore CM5 sensor chip compared to the amount ofwild-type FVIIa bound subsequent to a standardized injection time andprotease concentration.

For every experiment, soluble TFPI (R&D Systems) was immobilized to anew 4-flow cell Biacore CM5 Series S sensor chip (GE Healthcare) usingthe amine coupling protocol available within the Biacore T-100 controlSoftware (GE Healthcare) and the reagents provided with the AmineCoupling Kit (GE Healthcare). All four available flow cells wereutilized for immobilization of two different densities of TFPI andbovine serum albumin (BSA), which served as a blocking agent in thereference cells. BSA was diluted to 5 μg/mL in sodium acetate (pH 4.0)and immobilized in flow-cells 1 and 3 at 1000 and 2000 response units(RU), respectively. For TFPI immobilization, lyophilized soluble TFPI(10 μg) was resuspended in 100 μL of 1× Coupling Buffer (30 mM Hepes,135 mM NaCl, 1 mM EDTA, 0.01% Tween-20, pH 7.4) to a concentration of0.1 mg/mL. A total of 20 μL of 0.1 mg/mL TFPI as diluted to 10 μg/mL insodium acetate pH 4.0 for immobilization to flow-cells 2 and 4 at 1000and 2000 RU, respectively. Coupling buffer was used as the runningbuffer during the immobilization steps.

Each sample of FVIIa was prepared at a final concentration of 320 nM in1× Running Buffer (20 mM Hepes, 150 mM NaCl, 5 mM CaCl₂, 0.1% PEG 8000,0.1% BSA, 0.01% Tween-20, pH 7.4) containing 620 nM sTF (HumanCoagulation Factor III; R&D Systems). Generally, each FVIIa variant wasdiluted 10-fold into 1× Running Buffer before the final dilution of 320nM. FVIIa/sTF complexes were prepared at a final volume of 120 μL induplicate allowing for up to 48 unique FVIIa variants to be loaded intoa 96-well storage plate and evaluated with duplicate injections in asingle run. The FVIIa/sTF complex was incubated at RT for 10-15 minbefore initiation of the first sample injection.

A standardized binding analysis method was created within the BiacoreControl Software (GE Healthcare) in which every FVIIa replicate isinjected for 180 seconds of association time followed by a short 60seconds of dissociation at a flow rate of 10 μL/min. Regeneration of thesensor chip followed the dissociation phase for 30 seconds with 10 mMglycine, 500 mM NaCl, pH 3.0 and then a 60 second stabilization periodwith 1× Running Buffer at the same 10 μL/min flow rate. Two assayreference points were recorded for each run and subsequent dataanalysis, one 5 seconds prior to the conclusion of the association phase(binding) and a second reported 5 seconds before the conclusion of thedissociation phase (dissociation). Before initiating a full assay, thesensor chip was tested with a single injection of 320 nM wild-typeFVIIa/sTF for 180 seconds, which should give a response of approximately400-450 RU and 750-850 RU for binding to flow-cells 2 (1000 RU) and 4(200 RU), respectively.

Data analysis was performed first with the Biacore T100 EvaluationSoftware (GE Healthcare) to inspect the assay validation parameters,which include verifying that binding to the reference cell is minimal,baseline drift and the binding of control blank injections (runningbuffer). Data tables were generated within this application thatindicated the amount of FVIIa variant bound (in RU) at both the bindingreport point and the dissociation report point. The data tables weresubsequently exported for further analysis within the Microsoft Excelspreadsheet environment. The raw data points (RU bound) were correctedfor control binding to the sensor chip and then a ratio of the amount ofwild-type FVIIa bound (in RU) to the amount of FVIIa variant bound (inRU) was taken for each parameter and reported as Binding (wt/variant)and Dissociation (wt/variant). Table 22 present the results of thestudy. Resistance to TFPI inhibition is reflected as an increase in theratio for one or both of the evaluated parameters. For instance, aBinding (wt/variant) or Dissociation (wt/variant) value of 20 for aparticular FVIIa variant indicates that that variant is 20-fold moreresistant to TFPI inhibition than wild-type FVIIa. Several variantsexhibited increased resistance to TFPI inhibition. For example, variantscontaining the K341D mutation (mature FVII numbering) such asQ286R/M298Q/K341D-FVIIa, Q286R/K341D-FVIIa and M298Q/K341D-FVIIa, haveratios indicating significant resistance to TFPI (greater than40-150-fold). In some cases, the rate of dissociation was affected morethan the rate of association.

TABLE 22 Resistance of FVIIa variants to inhibition by TFPI TF-DependentTFPI Resistance Assay 293-F Cells BHK-21 Cells Binding DissociationBinding Dissociation Mutation (mature FVII Mutation (Chymotrypsin (wt/(wt/ (wt/ (wt/ Numbering) Numbering) variant) variant) variant) variant)WT WT 1.0 1.0 1.0 1.0 Q286R Q143R 2.2 1.9 3.7 3.4 A292N/A294SA150N/A152S 2.6 2.2 A175S A39S 1.6 1.4 K109N K[109]N 0.8 0.7 A122N/G124SA[122]N/G[124]S 1.2 1.1 A122N/G124S/ A[122]N/G[124]S/E245N/P246A/ 1.11.0 E394N/P395A/R396S R247S S119N/L121S S[119]N/L[121]S 1.2 1.3T128N/P129A T[128]N/P[129]A 1.1 1.2 Q286R/S222A/Gla Swap Q143R/S82A/Glaswap FIX 1.9 1.7 FIX Q286R/M298Q Q143R/M156Q 2.0 1.8 Q286R/M298Q/K341QQ143R/M156Q/K192Q 3.1 3.5 3.0 3.5 Q286R/M298Q/K199E Q143R/M156Q/K60cE4.6 4.1 2.4 2.1 T239V T99V 1.4 1.4 1.1 1.1 H257A/M298Q H117A/M156Q 2.42.7 S222A/H257A/Q286R/M298Q S82A/H117A/Q143R/M156Q 1.6 1.4A175S/Q286R/Q366V A39S/Q143R/Q217V 4.2 3.5 3.5 2.9 K109N/A175SK[109]N/A39S 3.4 3.2 S222A/Q286R/Q366V S82A/Q143R/Q217V 1.2 1.2 Q3661Q2171 0.6 0.5 K197E/M298Q K60aE/M156Q 2.6 2.3 Q286R/M298Q/K341DQ143R/M156Q/K192D 33.6 182.3 Q286R/K341D Q143R/K192D 29.8 281.8M298Q/K341D M156Q/K192D 146.8 196.5 S119N/L121S/A175SS[119]N/L[121]S/A39S 2.8 2.7 T128N/P129A/A175S T[128]N/P[129]A/A39S 2.72.6 A122N/G124S/A175S A[122]N/G[124]S/A39S 3.0 2.9

Example 9 Pharmacokinetic Analysis of FVIIa Polypeptides

Pharmacokinetic properties of FVIIa polypeptides were assessed bymeasuring the amount of human Factor VIIa in mouse plasma. Two assayscan be used to quantify FVIIa in plasma. An ELISA can be used toquantify total FVIIa protein in mouse plasma and a FVIIa-dependantclotting assay (FVIIa:C) used to quantify coagulant activity of theFVIIa polypeptides in plasma.

A. Administration of FVIIa Polypeptides to Mice

Modified FVIIa polypeptides and the unmodified recombinant human FVIIa(rhFVIIa) protein (NovoSeven®, Novo Nordisk) were evaluated inpharmacokinetic studies. For each study, 18 male CD-1 mice were injectedwith an intravenous bolus dose (0.1-3.0 mg/kg, depending on the study)of rFVIIa. At 5, 15, 30, 60, 120, and 240 minutes post-injection, threemice from each injection protocol were euthanized using CO₂ asphyxiationand approximately 1.0 mL of blood was drawn into a 1.0 mL syringe via apercutaneous cardiac puncture. Each syringe was pre-loaded withsufficient sodium citrate to achieve a final concentration of 3.2% in 1mL of blood. The blood samples were then centrifuged at 9000 rpm for 8min at 4° C. The plasma was removed to labeled individual 1.5 ml tubes(Eppendorf), snap frozen in liquid nitrogen and stored at −80° C.Additionally, one mouse per experiment was injected with vehicle alone(sham) and plasma from this mouse was used for background FVIIa activitydetermination.

B. ELISA Assay

A commercially available kit, IMUBIND® Factor VII ELISA (AmericanDiagnostica) was used to detect FVII protein in serum by ELISA. This kitemploys a plate pre-coated with an anti-FVII/FVIIa antibody to capturethe protein, and a biotinylated anti-FVII antibody for detection througha steptavidin labeled horseradish peroxidase. The kit was used accordingto the manufacturers' direction with the following exceptions: first,the standard curve has been narrowed to ensure a linear range over theentire concentration range and spans the concentrations of 0.88 ng/ml to10 ng/ml; second, the purified FVIIa variant itself was used for thestandard curve rather than the FVII standard provided with the kitbecause of differences in the antibody affinity. Experiments indicatedthat the complex of FVIIa with anti-thrombin III (ATIII), a potentialplasma inhibitor of FVIIa, is detected at 75% of the level of the freeprotease, ensuring that the assay can detect the total FVIIa in theplasma sample in both the active and inactive forms.

Briefly, the plasma samples were thawed at room temperature and diluted10 fold in sample buffer (PBS+0.1% Triton X-100+1.0% BSA) then dilutedserially 5.5 fold for four dilutions. The four diluted samples werediluted 2 fold onto the ELISA plate for final sample dilutions of 20,110, 605 and 3327.5 fold. These four dilutions of mouse plasma covered arange of protease concentrations from 33,000 ng/ml to 20 ng/ml. Eachsample was measured on two separate assays plates, and thosemeasurements within the range of the standard curve were used tocalculate the concentration of FVIIa variant in the plasma sample

C. Clotting Assay

A commercially available kit (STACLOT FVIIa-rTF, Diagnostica Stago,Parsippany, N.J.) was used as the clotting assay. To determine thecoagulant activity of the active FVIIa polypeptides, plasma samples wereassayed using a FVIIa-dependant clotting assay (FVIIa:C). The assay wasperformed using reagents and instructions provided in a commercial kitand clotting time measured using an electromechanical clot detectioninstrument (STArt4, Diagnostica Stago, Parsippany, N.J.). The kit wasused according to the manufacturers' direction with the followingexceptions: first, the purified FVIIa variant itself was used for thestandard curve rather than the rhFVIIa standard provided with the kit;second, the following bulk commercial reagents were used for routinepharmacokinetic screening studies and gave comparable results to the kitreagents: soluble tissue factor (CalBioChem, La Jolla, Calif.) andsynthetic phospholipid blend (Avanti Polar Lipids, Alabaster, Ala.),TBSA buffer (Tris-NaCl, pH 7.5 with 1% BSA; DiaPharma, West Chester,Ohio), and 25 μM calcium chloride solution (Diagnostica Stago,Parsippany, N.J.).

The clotting assay was performed as follows. Frozen plasma samples werethawed at room temperature for approximately 45 min and then diluted1:5000 in buffer. Fifty μl of the diluted plasma is combined with 50 μLFactor VII-deficient human plasma and 50 μL of relipidated tissue factorand pre incubated for 180 seconds. Following preincubation, 50 μL ofcalcium chloride solution (25 μM) was added to initiate clotting.Clotting time was determined using electromechanical clot detection.Each plasma sample was assayed in duplicate. The system was calibratedby constructing a standard curve using the clotting time of serialdilutions of buffer containing a known amount of the specific FVIIavariant being assayed. FVIIa concentrations in mouse plasma samples werecalculated from the linear portion of the log FVIIa versus Log clottingtime standard curve. The ability of plasma samples to induce clotting inFactor VII-deficient plasma was reported as ng FVIIa/mL of mouse plasmafollowing background subtraction of endogenous wild type FVIIa in plasmafrom sham treated mice.

The half-life of each FVII protein was routinely determined by making aconventional fit of the natural log of the activity to a straight line,and measuring the time taken for the activity of FVIIa proteins to bereduced by half. For FVII proteins with multi exponential decay,half-life was determined from the terminal portion of the log plasma VStime profile. Additional pharmacokinetic parameters were calculated asfollows: Plasma AUC_(0-inf)/Dose (calculated as [AUC_((0-t))+Ct/(ln2/T_(1/2))], where t is the last time point with measurable plasmaconcentration of the FVIIa polypeptide divided by the IV dose (mg/kg));half-life (the half life of the FVIIa polypeptide during the terminalphase of plasma FVIIa concentration-versus-time profile; T_(1/2) iscalculated as −ln 2 divided by the negative slope during the terminalphase of the log-linear plot of the plasma FVIIaconcentration-versus-time curve); MRT_(0-last) (mean time the FVIIapolypeptide resides in body; calculated as AUMC_(0-last)/AUC_(0-last),where AUMC_(0-last) is the total area under the first moment-versus-timecurve (FVIIa concentration time versus time curve), and is calculated bythe linear trapezoid rule); Cl (systemic clearance; calculated asDose/AUC_(0-inf)); and V_(d) (volume of distribution based on theterminal elimination e constant (β); calculated as [Cl/(ln 2/T_(1/2))]).

D. Pharmacokinetic Properties of FVIIa Variants

Using the above described FVIIa:C protocol, the pharmacokineticproperties of wild-type FVIIa and FVIIa variants were assessed based onclotting activity in plasma. The results are set forth in Table 23.Several FVIIa variants exhibited improved pharmacokinetic parameterscompared to wild-type FVIIa.

TABLE 23 Mouse Pharmacokinetic Parameters of FVIIa Variants PlasmaAUC0-inf Half- MRT0- Cl Vd Mutation (mature FVII IV Dose (ug · min/mL)/Life last (mL/min/ (mL/ numbering) (mg/kg) Dose (min) (min) kg) kg) NNovoSeven ® RT FVIIa 0.1 1165 37 50 0.9 46 1 NovoSeven ® FVIIa 0.1 74130 31 1.4 58 1 NovoSeven ® FVIIa 1.0 1156 36 41 1.7 89 1 WT 0.1 686 3737 1.8 92 6 WT 1.0 798 50 57 1.5 118 2 P257insGGGSCSFGRGDIRNVC 0.1 39132 32 2.3 108 1 T128N/P129A 0.1 1392 57 43 0.8 64 2 S52A 0.1 594 51 581.6 118 1 K109N 0.1 951 50 40 1.1 75 1 A51N 0.1 270 33 32 3.7 174 1S52A/S60A 0.05 460 25 29 2.2 78 1 S52A/S60A 0.1 408 17 34 2.5 61 1 M298Q0.1 258 76 33 4.9 443 2 T128N/P129A/M298Q 0.1 495 26 28 2.0 75 1V158D/E296V/M298Q 0.05 72 13 10 13.9 258 1 V158D/E296V/M298Q 0.1 4416(á), 20 22.8 1465 1 45(â) V158D/E296V/M298Q 1.0 39 7 13 26.6 979 2V158D/E296V/M298Q 3.0 22 7.7á 7.9 45.6 506 1 V158D/E296V/M298Q 6.0 48 4345 20.8 1305 1 T128N/P129A/V158D/E296V/ 0.1 125 11 14 8.0 122 1 M298QS52A/S60A/V158D/E296V/M298Q 0.1 47 15 11 21.4 450 1 Q286R 0.1 181 55 595.5 439 1 Q286R 0.3 460 41.7 76 1.8 110 1 Q286R 1.0 1256 67 68 0.8 80 2T128N/P129A/Q286R 0.1 2443 96 82 0.4 56 1 S52A/S60A/Q286R 0.1 1565 43 600.6 55 1 Gla Swap FIX 0.1 207 20 22 4.9 140 2 K341D 0.1 2817 106 114 0.122 1 S222A 0.1 189 32 33 5.3 248 1 S222A 1.0 139 30 33 3.8 293 1T128N/P129A/S222A 0.1 422 28.4(á), 58 2.4 244 1 71(â) S52A/S60A/S222A0.1 270 45 38 3.7 243 1 H257A 0.1 595 31 30 1.7 76 1 H257S 0.1 1015 5962 1.0 84 1 H257S 0.75 816 50 62 1.2 85 1 Q366V 0.1 19 14 11 51.6 1060 1Gla SwapFIX/Q366V 0.1 157 11 7.0 6.4 107 1 A122N/G124S/E394N/P395A/ 0.11089 63 45 0.9 84 1 R396S G318N 1.0 568 95 56 1.8 242 1 A175S 0.1 2081111 85 0.5 76 2 K109N/A175S 0.1 1280 183 51 0.8 207 1 S119N/L121S/A175S0.1 2460 102 50 0.4 60 1 T128N/P129A/A175S 0.1 2770 83 48 0.4 43 1A122N/G124S/A175S 0.1 3240 91 50 0.3 40 1 A122N/G124S 0.1 679 52.9 421.1 87 1 H257A/Q286R 0.1 1848 56.8 73 0.5 44 1 S222A/Q286R 0.1 861 30 381.5 97 2 Gla SwapFIX/S222A/Q286R 1.0 415 39 42 2.4 136 1 Gla SwapFIX/T128N/P129A/ 0.1 929 55 59 1.1 86 1 S222A/Q286R S222A/H257A/Q286R0.1 976 56 42 1.0 83 1 Q286R/M298Q 0.055 1198 40 44 0.8 49 1 Q286R/M298Q0.1 605 35 42 1.9 94 6 Q286R/M298Q 1.0 363 29 36 2.8 116 1 Gla SwapFIX/Q286R/M298Q 0.1 231 26 20 4.9 194 3 T128N/P129A/Q286R/M298Q 0.1 157141 60 0.6 38 1 T[128]N/P[129]A/ 0.1 1326 42.3 50 0.8 47 1 Q286R/M298QGla Swap FIX/T128N/P129A/ 0.1 427 30 32 2.3 101 1 Q286R/M298Q {Gla SwapFIX/E[40]L}/ 0.1 386 53 43 2.6 198 1 Q286R/M298Q {Gla Swap FIX/K[43]I}/0.1 439 85 47 2.3 280 1 Q286R/M298Q {Gla Swap FIX/Q[44]S}/ 0.1 291 14 243.4 150 1 Q286R/M298Q {Gla Swap FIX/M[19]K}/ 0.1 648 14(á), 43 1.5 32 1Q286R/M298Q 75(â) S52A/S60A/Q286R/M298Q 0.1 374 32 34 2.7 122 1 Gla SwapFIX/S52A/S60A/ 0.1 256 19 18 3.9 107 1 Q286R/M298Q {Gla SwapFIX/K[43]I}/ 0.1 154 18 17 6.5 166 1 T128N/P129A/Q286R/M298Q S222A/M298Q0.1 72 17 12 13.9 332 1 S222A/H257A/Q286R/M298Q 0.1 93 33 30 10.8 517 1T128N/P129A/S222A/H257A/ 0.1 770 38 45 1.3 72 1 Q286R/M2986QS52A/S60A/S222A/H257A/Q28R/ 0.1 469 26 28 2.1 78 1 M2986QA1755/Q286R/Q366V 0.1 353 28.9 23 2.8 118 1 Q286R/M298Q/K341D 0.1 219 2320 4.6 151 1 M298Q/K341D 0.1 2430 78 76 0.4 46 1 Q286R/M298Q/Q366N 0.1569 29 32 1.8 74 1 T128N/P129A/Q286R/M298Q/ 0.1 1897 50 58 0.5 38 1Q366N {Gla Swap FIX 0.1 257 10(á), 20 3.9 163 1K[43]I}/Q286R/M298Q/Q366N 28(â) {Gla Swap FIX K[43]I}/ 0.1 393 24 27 2.589 1 T128N/P129A/Q286R/M298Q/ Q366N T128N/P129A/Q286R/H373F 0.1 1467 5962 0.9 74 1 Q286R/M298Q/H373F 0.1 466 28 23 2.2 87 1T128N/P129A/Q286R/M298Q/ 0.1 597 27(á), 51 1.6 153 1 H373F 67(â)T128N/P129A/M298Q/H373F 0.1 307 7(á), 23 3.3 126 1 27(â)V158D/Q286R/E296V/M298Q 0.1 172 14 36 5.8 535 1 S222A/T239V 0.1 127 4739 7.9 535 1 Gla Swap FIX/S222A/ 0.1 460 34 33 2.2 108 1 T239V/Q286RT239V/Q286R/M298Q 0.1 398 28(á), 60 2.5 258 1 71(â) Gla Swap FIX/T239V/0.1 365 13(á), 29 2.7 220 1 Q286R/M298Q 56(â) T128N/P129A/T239V/Q286R/0.1 914 38 36 1.1 60 1 M298Q S222A/T239V/H257A/Q286R/ 0.1 181 28 31 5.5225 1 M298Q T128N/P129A/S222A/T239V/ 0.1 564 27 30 1.8 70 1H257A/Q286R/M298Q T239V/Q286R/H373F 0.1 385 72 54 2.6 269 1T239V/Q286R/M298Q/H373F 0.1 149 36 23 6.7 353 1 T128N/P129A/T239V/Q286R/0.1 345 27 27 2.9 113 1 M298Q/H373F V158D/T239I/E296V/M298Q 0.1 37014(á), 50 2.7 273 1 70(â) T239I/Q286R 0.1 1820 85 76 0.6 68 1S222A/T239I 0.1 1300 6(á), 69 0.8 90 1 81(â) Gla Swap FIX/ 0.1 1073 7066 0.9 94 1 S222A/T239I/Q286R T239I/Q286R/M298Q 0.1 1029 27(á), 62 1.084 1 60(â) Gla Swap FIX/ 0.1 1269 54 62 0.8 61 1 T239I/Q286R/M298QT128N/P129A/T239I/Q286R/ 0.1 2105 82 74 0.5 56 1 M298QS222A/T239I/H257A/Q286R/ 0.1 1212 31(á), 60 0.8 101 1 M298Q 79(â)T239I/Q286R/H373F 0.1 1841 30(á), 69 0.5 62 1 85(â) V158D/T239V/E296V/0.1 184 24(á), 78 5.4 1053 1 M296Q 134(â) T239V/Q286R 0.1 1522 29(á), 680.7 68 1 72(â) T239V/Q286R/M298Q/H373F 0.1 950 36 61 1.1 55 1T239V/Q286R/M298Q/H373F 0.1 806 32 56 1.3 57 2 T239V/Q286R/M298Q/H373F0.1 663 27 52 1.5 59 1 T239V/Q286R/M298Q/H373F 0.1 1350 53 62 0.7 57 1S222A/H257S/Q286R/M298Q 0.1 814 33 31 1.2 59 1 H257S/Q286R/M298Q/H373F0.1 297 34 34 3.4 163 1 S222A/Q286R/M298Q/H373F 0.1 106 35 23 9.4 478 1Gla Swap FIX/S222A/ 0.1 104 9.1(á), 13 9.7 242 1 Q286R/M298Q/H373F 17(â)S222A/Q286R/M298Q 0.1 347 24 26 2.9 102 1 Gla Swap FIX/S222A/ 0.1 263 1113 3.8 62 1 Q286R/M298Q T128N/P129A/A175S/Q366V 0.1 2196 52(á), 78 0.556 1 85(â) A122N/G124S/A175S/Q366V 0.1 2148 92 81 0.5 62 1T128N/P129A/A175S/S222A 0.1 4248 122 88 0.2 41 1 A122N/G124S/A175S/S222A0.1 3316 102 83 0.3 44 1 T128N/P129A/A175S/Q286R 0.1 6160 151 94 0.2 351 A122N/G124S/A175S/Q286R 0.1 4097 139 93 0.2 49 1 Gla Swap FIX/ 0.1 48026 30 2.1 79 1 S222A/Q286R/H373F V258D/E296V/M298Q/H373F 0.1 90 8.7(á),14 11.1 321 1 20(â) H257A/Q286R/M298Q 0.1 1029 42 48 1.0 59 1 Gla SwapFIX/T128N/ 0.1 2787 38(á), 88 0.4 69 1 P129A/A175S/S222A/Q286R 134(â)Gla Swap FIX/A122N/ 0.1 3492 148 95 0.3 61 1 G124S/A175S/S222A/Q286RT128N/P129A/A175S/Q286R/ 0.1 5120 171 96 0.2 48 1 M298Q A122N/G124S/ 0.13681 154 92 0.3 61 1 A175S/Q286R/M298Q T128N/P129A/A175S/S222A/ 0.1 3140113 87 0.3 52 1 H257A/Q286R/M298Q A122N/G124S/A175S/S222A/ 0.1 265937.(á), 85 0.4 70 1 H257A/Q286R/M298Q 130(â) T128N/P129A/A175S/Q286R/0.1 3580 118 88 0.3 48 1 M298Q/H373F A122N/G124S/A175S/Q286R/ 0.1 3148105 84 0.3 48 1 M298Q/H373F V158D/Q286R/E296V/M298Q/ 0.1 124 20 17 8.1237 1 H373F M298Q/H373F/Q366N 0.1 169 8 8.2 5.9 69 1 T239V/M298Q/H373F0.1 110 18 16 9.1 235 1 T239I/M298Q/H373F 0.1 562 26 28 1.8 66 1T128N/P129A/Q286R/M298Q/ 0.1 607 29 30 1.6 69 1 Q366N/H373FT239V/Q286R/M298Q/ 0.1 729 29 30 1.4 57 1 Q366N T239I/Q286R/M298Q/Q366N0.1 1548 72 73 0.6 67 1 α = alpha half life, measuring distribution halflife β = beta half life, measuring elimination half life

Table 24 sets forth the results of the study using the followingpharmacokinetic parameters: % Recovery (in vivo)(the measured plasmaconcentration of FVIIa at 5 minutes post-dose (first time point) dividedby the theoretical maximum FVIIa plasma concentration (based onadministered FVIIa mass and theoretical total blood volume) times 100%);% Recovery (in vitro)(the measured FVIIa concentration in plasma spikedwith a known amount of FVIIa divided by the theoretical FVIIa plasmaconcentration (based on amount of FVIIa mass spiked into a known plasmavolume) times 100%); AUC*Activity/Dose (TF-Dependent) (the plasmaAUC/Dose multiplied by the TF-Dependent Indirect Activity (see Table 15,above); Improvement in Activity Exposure over NovoSeven® FVIIa(TF-Dependent) (calculated by AUC*Activity/Dose(TF-Dependent)_(NovoSeven® FVIIa)/AUC*Activity/Dose(TF-Dependent)_(Mutant FVIIa)); AUC*Activity/Dose (TF-Independent) (theplasma AUC/Dose multiplied by the TF-Independent Indirect Activity (seeTable 15, above); Improvement in Activity Exposure over NovoSeven® FVIIa(TF-Independent) (calculated by AUC*Activity/Dose(TF-Independent)_(Novoseven® FVIIa)/AUC*Activity/Dose(TF-Independent)_(Mutant FVIIa)).

TABLE 24 Improvement Improvement in Activity in Activity Exposure overAUC * Activity/ Exposure over % AUC * Activity/ Novo7 Dose Novo7Mutation (mature Recovery Dose (TF- (TF- (TF- (TF- FVII numbering) (invivo) Dependent) Dependent) Independent) Independent) NovoSeven ® FVIIa60% 2.95E+10 1.0 7.26E+03 1.0 WT 46% 4.71E+10 1.4 1.22E+04 1.5T239I/Q286R/M298Q/ 52% 1.15E+11 3.9 1.38E+05 18.9 Q366N T239V/Q286R/ 66%1.35E+11 4.6 4.69E+04 6.5 M298Q/Q366N T128N/P129A/Q286R/ 66% 8.10E+102.8 9.77E+04 13.5 M298Q/Q366N/ H373F T239I/M298Q/H373F 60% 1.95E+10 0.77.77E+04 10.7 T239V/M298Q/H373F 17% 4.89E+09 0.2 3.53E+04 4.9M298Q/H373F/Q366N 32% 1.19E+10 0.4 2.13E+04 2.9 V158D/Q286R/E296V/ 21%2.97E+10 1.0 7.12E+04 9.8 M298Q/H373F A122N/G124S/A175S/ 79% 2.64E+119.0 ND ND Q286R/M298Q/H373F T128N/P129A/A175S/ 85% 2.05E+11 7.0 ND NDQ286R/M298Q/H373F A122N/G124S/A175S/ 76% 2.23E+11 7.6 ND ND S222A/H257A/Q286R/M298Q T128N/P129A/A175S/ 69% 2.16E+11 7.4 6.36E+04 8.8S222A/H257A/Q286R/ M298Q A122N/G124S/ 84% 2.73E+11 9.3 ND NDA175S/Q286R/M298Q T128N/P129A/A175S/ 76% 3.69E+11 12.6 ND ND Q286R/M298QGla Swap FIX/ 42% 1.44E+11 4.9 ND ND A122N/G124S/ A175S/S222A/Q286R GlaSwap FIX/ 69% 1.08E+11 3.7 8.51E+04 11.7 T128N/P129A/ A175S/S222A/Q286RH257A/Q286R/ 42% 1.16E+11 4.0 6.33E+04 8.7 M298Q V258D/E296V/ 17%1.36E+10 0.5 1.17E+05 16.1 M298Q/H373F Gla Swap FIX/ 42% 5.88E+10 2.03.90E+04 5.4 S222A/Q286R/H373F A122N/G124S/A175S/ 78% 1.68E+11 5.81.22E+04 1.7 Q286R T128N/P129A/A175S/ 34% 2.05E+11 7.0 2.01E+04 2.8Q286R A122N/G124S/A175S/ 60% 6.90E+10 2.4 ND ND S222A T128N/P129A/A175S/88% 7.31E+10 2.5 8.00E+03 1.1 S222A A122N/G124S/A175S/ 60% 6.48E+10 2.26.42E+03 0.9 Q366V T128N/P129A/A175S/ 74% 7.43E+10 2.5 7.39E+03 1.0Q366V Gla Swap FIX/ 40% 5.52E+10 1.9 1.09E+05 15.0 S222A/Q286R/M298QS222A/Q286R/M298Q 22% 4.46E+10 1.5 8.78E+04 12.1 Gla Swap FIX/ 16%2.94E+10 1.0 1.99E+04 2.7 S222A/Q286R/M298Q/ H373F S222A/Q286R/M298Q/ 5%1.57E+10 0.5 4.57E+04 6.3 H373F H257S/Q286R/M298Q/ 20% 4.52E+10 1.59.01E+03 1.2 H373F S222A/H257S/Q286R/ 72% 1.28E+11 4.4 3.39E+04 4.7M298Q T239I/Q286R/M298Q/ 56% 8.73E+10 3.0 1.05E+04 1.5 H373F T239V/Q286R66% 1.35E+11 4.6 1.79E+04 2.5 V158D/T239V/E296V/ 8% 4.08E+10 1.43.43E+05 47.3 M296Q T239I/Q286R/H373F 77% 1.14E+11 3.9 ND NDS222A/T239I/H257A/ 61% 1.38E+11 4.7 6.26E+04 8.6 Q286R/M298QT128N/P129A/T239I/ 81% 1.72E+11 5.9 1.64E+05 22.6 Q286R/M298Q Gla SwapFIX/ 56% 1.59E+11 5.4 3.10E+05 42.6 T239I/Q286R/M298Q T239I/Q286R/M298Q54% 1.16E+11 4.0 1.16E+04 1.6 Gla Swap FIX/ 42% 7.27E+10 2.5 2.18E+043.0 S222A/T239I/Q286R S222A/T239I 71% 3.97E+10 1.4 2.19E+03 0.3T239I/Q286R 56% 1.05E+11 3.6 8.34E+03 1.1 V158D/T239I/E296V/ 34%5.38E+10 1.8 8.01E+04 11.0 M298Q T239V/Q286R/M298Q/ 10% 2.54E+10 0.96.07E+03 0.8 H373F T239V/Q286R/H373F 16% 4.09E+10 1.4 4.69E+03 0.6T128N/P129A/S222A/ 49% 6.84E+10 2.3 1.01E+05 13.9 T239V/H257A/Q286R/M298Q S222A/T239V/H257A/ 15% 3.87E+10 1.3 2.59E+04 3.6 Q286R/M298QT128N/P129A/T239V/ 60% 9.47E+10 3.2 2.10E+05 28.9 Q286R/M298Q (Gla Swap34% 9.24E+10 3.2 1.01E+05 14.0 FIX/T239V/ Q286R/M298Q T239V/Q286R/ 21%6.85E+10 2.3 1.17E+04 1.6 M298Q Gla Swap FIX/S222A/ 33% 9.32E+10 3.27.99E+03 1.1 T239V/Q286R S222A/T239V 10% 9.52E+09 0.3 3.83E+02 0.1V158D/Q286R/E296V/ 15% 2.56E+10 0.9 1.76E+05 24.3 M298QT128N/P129A/Q286R/ 36% 7.92E+10 2.7 1.83E+04 2.5 M298Q/H373FQ286R/M298Q/H373F 66% 1.39E+11 3.9 1.42E+05 12.1 T128N/P129A/Q286R/ 49%2.03E+11 6.9 1.93E+04 2.7 H373F {Gla Swap FIX 45% 9.88E+10 3.4 8.58E+0411.8 K[43]I}/T128N/ P129A Q286R/M298Q/ Q366N {Gla Swap FIX 36% 4.35E+101.5 2.18E+04 3.0 K[43]I}/Q286R/ M298Q/Q366N Q286R/M298Q/ 41% 1.33E+114.5 7.50E+04 10.3 Q366N M298Q/K341D 84% 4.26E+10 1.0 1.02E+04 0.6Q286R/M298Q/ 33% 4.98E+09 0.1 5.24E+03 0.3 K341D A175S/Q286R/Q366V 22%3.72E+10 0.9 7.31E+03 0.4 S52A/S60A/S222A/H257A/ 45% 1.17E+11 4.05.42E+04 7.5 Q28R/M2986Q T128N/P129A/S222A/ 53% 1.80E+11 6.1 5.23E+047.2 H257A/Q286R/ M2986Q S222A/H257A/Q286R/ 9% 3.04E+10 0.9 4.17E+04 4.9M298Q S222A/M298Q 7% 8.63E+09 0.2 9.51E+04 5.1 {Gla Swap 23% 2.46E+080.0 6.00E+04 8.3 FIX/K[43]I}/T128N/ P129A/Q286R/M298Q Gla Swap 32%4.63E+10 1.6 2.54E+05 34.9 FIX/S52A/S60A/ Q286R/M298Q S52A/S60A/ 31%4.67E+10 1.6 8.11E+04 11.2 Q286R/M298Q {Gla Swap 69% 9.11E+10 3.13.32E+05 45.7 FIX/M[19]K}/ Q286R/M298Q {Gla Swap 35% 5.77E+10 2.02.12E+05 29.2 FIX/Q[44]S}/ Q286R/M298Q {Gla Swap 28% 1.20E+11 4.13.96E+05 54.5 FIX/K[43]I}/ Q286R/M298Q {Gla Swap 36% 8.04E+10 2.73.14E+05 43.3 FIX/E[40]L}/ Q286R/M298Q Gla Swap FIX/ 84% 9.00E+10 3.13.58E+04 4.9 T128N/P129A/ Q286R/M298Q T[128]N/P[129]A/ 66% 1.55E+11 5.32.20E+05 30.3 Q286R/M298Q T[128]N/P[129]A/ 62% 4.21E+11 14.4 1.84E+0525.4 Q286R/M298Q Q143R/M156Q/Gla 21% 8.53E+10 2.7 2.30E+05 29.9 swap FIXQ286R/M298Q 51% 1.08E+11 3.2 1.03E+05 11.2 S222A/H257A/Q286R 40%1.55E+11 4.4 2.72E+04 3.2 Gla Swap 44% 1.37E+11 4.7 1.44E+04 2.0FIX/T128N/P129A/ S222A/Q286R Gla SwapFIX/S222A/ 32% 5.95E+10 1.42.15E+04 1.2 Q286R S222A/Q286R 51% 1.85E+11 5.4 4.61E+04 2.5 H257A/Q286R63% 3.66E+11 12.5 1.59E+04 2.2 A122N/G124S/A175S 82% 8.70E+10 2.42.18E+04 1.2 T128N/P129A/A175S 80% 6.16E+10 1.7 ND ND S119N/L121S/A175S60% 8.68E+10 2.5 ND ND K109N/A175S 22% 4.73E+10 1.3 ND ND A175S 52%8.10E+10 2.4 1.40E+04 0.8 G318N 37% 5.22E+10 1.2 0.00E+00 0.0A122N/G124S/E394N/ 46% 1.07E+11 2.5 ND ND P395A/R396S Gla SwapFIX/Q366V26% 1.43E+10 0.5 1.41E+04 1.9 Q366V 3% 4.09E+09 0.1 1.44E+03 0.1 H257S39% 1.67E+11 5.1 1.30E+04 0.9 H257A 36% 4.96E+10 1.2 1.18E+04 0.6S52A/S60A/S222A 11% 1.05E+10 0.4 5.28E+03 0.7 T128N/P129A/S222A 21%5.28E+10 1.8 4.06E+03 0.6 S222A 10% 1.55E+10 0.4 8.56E+03 0.5 K341D 98%4.77E+10 1.1 ND ND S52A/S60A/Q286R 67% 6.42E+10 2.2 1.59E+04 2.2T128N/P129A/Q286R 67% 4.13E+11 14.1 4.24E+04 5.8 Q286R 35% 1.31E+11 3.73.52E+04 2.6 S52A/S60A/V158D/E296V/ 7% 9.34E+09 0.3 8.24E+04 11.3 M298QT128N/P129A/V158D/ 21% 5.03E+10 1.7 3.44E+05 47.4 E296V/M298QT128N/P129A/M298Q 58% 3.11E+10 1.1 4.49E+04 6.2 M286Q 18% 1.33E+10 0.44.93E+04 3.8 S52A/S60A 51% 5.36E+10 1.4 7.76E+03 0.6 A51N 15% 4.65E+101.1 9.39E+03 0.5 A122N/G124S 45% 5.26E+10 1.8 4.61E+03 0.6 A122N/G124S45% 9.20E+10 2.2 0.00E+00 0.0 K109N 52% 1.05E+11 3.6 ND ND K109N 52%8.43E+10 2.0 ND ND S52A 37% 3.50E+10 0.8 0.00E+00 0.0 Gla Swap FIX 31%2.63E+10 0.7 1.28E+04 1.4 T128N/P129A 88% 8.25E+10 2.4 25147.5 2.6P257insGGGSCSFGR 38% 6.29E+10 1.5 ND ND GDIRNVC

Example 10 Determination of Factor VIIa Binding to Soluble Tissue Factor

The ability of the FVIIa variants expressed from HEK 293 or BHK cells tobind soluble tissue factor (sTF) was assessed using Biacore surfaceplasmon resonance. The FVIIa variants are assessed through measurementof the binding profile at three protease concentrations in two duplicateexperiments, using two different levels of sTF bound to a Biacore CM5chip.

A new Series S CM5 sensor chip (GE Healthcare Cat #BR1006-68) wascoupled with bovine serum albumin and soluble tissue factor using aBiacore T100 instrument. Coupling was carried out using a BiacoreCoupling Buffer (30 mM Na Hepes pH 7.4, 135 mM NaCl, 1 mM EDTA, 0.01%Tween-20) with an Amine coupling kit (GE Healthcare Cat # BR-1000-50)and the protocol wizard in the Biacore T100 software. For theimmobilization, all four cells of the chip were used. Cells 1 and 3 werecoupled with 500 response units (RU) bovine serum albumin referenceprotein diluted in Acetate buffer, pH 4.0 and cells 2 and 4 were coupledwith 500 and 250 RU of sTF (R&D Systems) diluted in Acetate buffer, pH4.5.

Each FVIIa variant, and the wild-type FVIIa protease, was tested atthree concentrations and in duplicate. The proteases were diluted to 60nM, 30 nM and 15 nM in 100 μL Biacore Assay buffer (20 mM Na Hepes, pH7.4, 150 mM NaCl, 5 mM CaCl₂, 0.1% PEG 8000, 0.1% BSA, 0.01% Tween-20)in a 96 well assay plate. Each sample was assayed in the Biacore T100instrument using 120 seconds of contact time followed by 180 seconds ofdissociation time at a 10 μL/min flow rate. A buffer blank also wasassayed. The chip was regenerated with 50 mM EDTA, pH 7.0 for 60 secondsthen 30 seconds. The assay to measure binding of wild-type FVIIa to sTFshould yield three sets of curves that give a K_(d) of approximately 8nM.

Biacore T100 Evaluation software was used to analyze the data.Specifically, the Kinetics/Affinity 1:1 Binding analysis, which fits thedata to the Langmuir isotherm, was utilized and the data wasindividually fit for two replicates of each variant at two response unitcouplings. The four fit K_(d) values were averaged and are presented inTable 25. FVIIa variants containing the M298Q mutation tended to exhibitlower K_(d) results and thus bind more tightly to sTF.

TABLE 25 Binding of FVIIa variants to soluble TF Affinity K_(d) (nM)Mutation (mature FVII Mutation (chymotrypsin 293-F BHK numbering)numbering) cells cells wt wt 7.9 9.0 Q286N Q143N 8.9 Q286E Q143E 3.8Q286D Q143D 9.8 Q286S Q143S 8.2 Q286T Q143T 10.6 Q286R Q143R 7.6 Q286KQ143K 8.2 Q286A Q143A 6.3 Q286V Q143V 11.9 S222A S82A 4.2 H257S H117S4.2 Q366D Q217D 3.2 Q366E Q217E 5.6 Q366N Q217N 3.8 Q366T Q217T 7.1Q366S Q217S 9.0 Q366V Q217V 7.9 A175S A39S 6.5 V158T/L287T/M298KV21T/L144T/M156K 8.4 V158D/L287T/M298K V21D/L144T/M156K 8.5 Q286R/S222AQ143R/S82A 7.3 Q286R/S222A/Gla Swap Q143R/S82A/Gla swap 8.4 FIX FIXQ286R/M298Q Q143R/M156Q 4.7 Q286R/M298Q/K341Q Q143R/M156Q/K192Q 11.4Q286R/M298Q/K199E Q143R/M156Q/K60cE 4.9 S222A/M298Q S82A/M156Q 5.1H257A/M298Q H117A/M156Q 3.1 S222A/H257A/ S82A/H117A/Q143R/M156Q 2.5Q286R/M298Q Q286R/Q366V Q143R/Q217V 29.3 A175S/Q286R/Q366VA39S/Q143R/Q217V 18.3 S222A/Q286R/Q366V S82A/Q143R/Q217V 8.6 Q286M Q143M7.1 Q286L Q143L 7.1 Q286Y Q143Y 7.5 Q366I Q217I 6.7 Q366L Q217L 5.2Q366M Q217M 4.7 H216A/H257A H76A/H117A 6.0 Q286R/K341D Q143R/K192D 3.5M298Q/K341D Q143R/Q217D 7.9 Q286R/Q366N Q143R/Q217N 8.0Q286R/M298Q/Q366D Q143R/M156Q/Q217D 4.6 Q286R/M298Q/Q366NQ143R/M156Q/Q217N 3.8

An additional set of experiments was performed to assess the binding ofFVIIa variants to soluble TF using the same assay as described above,but with a modification of the FVIIa dose range to 30 nM, 15 nM and 7.5nM and the data analysis such that a two-state model was used to fit theSPR data. This two-state model analysis was provided in the Biacore T100Evaluation Software suite and reproduced below. The results are providedin Table 26, below.

TABLE 26 Binding of FVIIa variants to soluble TF Affinity Mutation(mature FVII Mutation (chymotrypsin Affinity K_(d-WT)/ numbering)numbering) K_(d) (nM) SD % CV K_(d-mut) n WT (NovoSeven ®) WT(NovoSeven ®) 6.5 1.2 19% 1.2 19 WT (NovoSeven-RT ®) WT (NovoSeven-RT ®)6.3 85% 13% 1.2 4 WT WT 7.6 1.9 24% 1.0 6 WT† WT† 3.9 0.6 15% 1.0 6T128N/P129A T[128]N/P[129]A 6.8 1.2 18% 1.1 7 Gla swap FIX Gla swap FIX36.0 2.6 7% 0.2 3 K109N K[109]N 10.8 0.2 2% 0.7 2 S52A/S60AS[52]A/S[60]A 23.2 4.0 17% 0.3 2 M298Q M156Q 5.9 0.7 11% 1.3 4 M298Q†M156Q† 1.9 0.1 7% 2.0 2 T128N/P129A/M298Q† T[128]N/P[129]A/M156Q† 2.40.4 16% 1.6 2 V158D/E296V/M298Q V21D/E154V/M156Q 2.0 0.5 26% 3.9 10V158D/E296V/M298Q† V21D/E154V/M156Q† 1.8 0.5 30% 2.2 4T128N/P129A/V158D/E296V/ T[128]N/P[129]A/V21D/E154V/ 2.0 0.1 4% 3.9 2M298Q M156Q S52A/S60A/V158D/E296V/ S[52]A/S[60]A/V21D/E154V/ 14.6 1.3 9%0.5 2 M1298Q M156Q Q286R Q143R 6.7 0.4 5% 1.1 2 T128N/P129A/Q286RT[128]N/P[129]A/Q143R 11.1 4.3 39% 0.7 6 T128N/P129A/Q286R†T[128]N/P[129]A/Q143R† 10.0 1.6 16% 0.4 4 S52A/S60A/Q286RS[52]A/S[60]A/Q143R† 51.6 18.4 36% 0.1 5 S222A S82A 2.6 0.1 4% 3.0 2T128N/P129A/S222A T[128]N/P[129]A/S82A 3.6 0.2 7% 2.1 2 S52A/S60A/S222AS[52]A/S[60]A/S82A 17.2 2.4 14% 0.4 2 H257S H117S 6.6 1.5 23% 1.2 2 GlaswapFIX/Q366V Gla swapFIX/Q217V 56.0 15.1 27% 0.1 3 A175S A39S 12.3 0.11% 0.6 2 K109N/A175S K[109]N/A39S 9.0 0.0 0% 0.8 2 S119N/L121S/A175SS[119]N/L[121]S/A39S 6.6 0.1 1% 1.1 2 T128N/P129A/A175ST[128]N/P[129]A/A39S 10.0 0.1 1% 0.8 2 A122N/G124S/A175SA[122]N/G[124]S/A39S 12.1 2.2 18% 0.6 2 Q286R/H257A Q143R/H117A 7.2 1.013% 1.1 2 Q286R/H257A† Q143R/H117A† 5.5 0.1 2% 0.7 2 Q286R/S222AQ143R/S82A 3.4 0.2 6% 2.3 2 Gla swap FIX/ Gla swap FIX/ 21.3 5.6 26% 0.46 T128N/P129A/ T[128]N/P[129]A/ S222A/Q286R S82A/Q143R Gla swap FIX/ Glaswap FIX/ 24.6 6.4 26% 0.2 6 T128N/P129A/ T[128]N/P[129]A/ S222A/Q286R†S82A/Q143R† Gla swap FIX/ Gla swap FIX/ 56.9 8.5 15% 0.1 2S52A/S60A/S222A/Q286R S[52]A/S[60]A/S82A/Q143R Q286R/S222A/H257AQ143R/S82A/H117A 6.0 0.5 8% 1.3 2 Q286R/M298Q Q143R/M156Q 4.1 0.5 11%1.9 9 Q286R/M298Q Q143R/M156Q† 3.6 1.1 29% 1.1 13 Q286R/M298QQ143R/M156Q§ 3.2 0.2 7% 1.2 4 Gla swap FIX/ Gla swap FIX/ 30.4 5.4 18%0.3 3 Q286R/M298Q Q143R/M156Q Gla swap FIX/ Gla swap FIX/ 22.1 1.5 7%0.2 8 Q286R/M298Q† Q143R/M156Q† T128N/P129A/Q286R/T[128]N/P[129]A/Q143R/ 4.7 1.2 26% 1.6 5 M298Q M156Q T128N/P129A/Q286R/T[128]N/P[129]A/Q143R/ 3.9 0.4 11% 1.0 8 M298Q† M156Q† Gla swap FIX/ Glaswap FIX/ 20.8 1.4 7% 0.4 5 T128N/P129A/Q286R/ T[128]N/P[129]A/Q143R/M298Q M156Q Gla swap FIX/ Gla swap FIX/ 37.5 12.3 33% 0.1 8T128N/P129A/Q286R/ T[128]N/P[129]A/Q143R/ M298Q† M156Q† {Gla swapFIX/E40L}/ {Gla swap FIX/E[40]L}/ 38.5 7.4 19% 0.2 2 Q286R/M298QQ143R/M156Q {Gla swap FIX/K43I}/ {Gla swap FIX/K[43]I}/ 35.3 3.4 10% 0.22 Q286R/M298Q Q143R/M156Q {Gla swap FIX/K43I}/ {Gla swap FIX/K[43]I}/23.7 3.6 15% 0.2 2 Q286R/M298Q† Q143R/M156Q† {Gla swap FIX/Q44S}/ {Glaswap FIX/Q[44]S}/ 40.8 6.4 16% 0.2 3 Q286R/M298Q Q143R/M156Q {Gla swapFIX/M19K}/ {Gla swap FIX/M[19]K}/ 16.7 2.4 14% 0.5 2 Q286R/M298QQ143R/M156Q S52A/S60A/Q286R/M298Q S[52]A/S[60]A/Q143R/M156Q 25.1 1.7 7%0.3 3 Gla swap FIX/ Gla swap FIX/ 6.0 0.1 1% 0.6 2S52A/S60A/Q286R/M298Q† S[52]A/S[60]A/Q143R/M156Q† {Gla swap FIX/ {Glaswap FIX/ 4.9 0.1 1% 1.6 2 M19K/E40L/K43I/Q44S}/M[19]K/E[40]L/K[43]I/Q[44] Q286R/M298Q S}/ Q143R/M156Q {Gla swapFIX/K43I}/ {Gla swap FIX/K[43]I}/ 10.5 1.3 13% 0.4 2 T128N/P129A/T[128]N/P[129]A/Q143R/M156Q† Q286R/M298Q† T239V T99V 5.2 1.5 1 T239IT99I 6.2 1.2 1 T128N/P129A/S222A/ T[128]N/P[129]A/S82A/H117A/ 2.6 0.311% 3.0 2 H257A/Q286R/M298Q Q143R/M156Q T128N/P129A/S222A/T[128]N/P[129]A/S82A/H117A/ 4.0 0.2 4% 1.0 2 H257A/Q286R/M298Q†Q143R/M156Q† S52A/S60A/S222A/H257A/Q286R/ S[52]A/S[60]A/S82A/H117A/ 11.12.7 24% 0.7 2 M298Q Q143R/M156Q T128N/P129A/Q286R/M298Q/T[129]N/P[129]A/Q143R/M156Q/ 3.2 0.6 18% 1.2 2 Q366N† Q217N† {Gla swap(Gla swap FIX/K[43]I}/ 13.0 0.7 5% 0.3 2 FIX/K43I}/Q286R/M298Q/Q366N†Q143R/M156Q/Q217N† {Gla swap {Gla swap FIX/K[43]I}/ 13.1 0.3 2% 0.3 2FIX/K43I}/T[128]N/P[129]A/ T[128]N/P[129]A/Q143R/M156Q/Q286R/M298Q/Q366N† Q217N† T128N/P129A/Q286R/H373FT[128]N/P[129]A/Q143R/H224F 6.8 0.0 0% 1.1 2 T128N/P129A/Q286R/M298Q/T[128]N/P[129]A/Q143R/M156Q/ 3.1 0.1 4% 2.4 2 H373F H224FS52A/S60A/Q286R/M298Q/ S[52]A/S[60]A/Q143R/M156Q/ 23.4 7.5 32% 0.3 3H373F H224F T128N/P129A/M298Q/H373F† T[128]N/P[129]A/M156Q/H224F† 1.30.5 41% 3.1 2 V21D/Q143R/E154V/M156Q V21D/Q143R/E154V/M156Q 2.2 0.2 8%3.5 2 Gla swap FIX/ Gla swap FIX/ 19.1 7.7 40% 0.4 7 S222A/T239V/Q286RS82A/T99V/Q143R Gla swap FIX/ Gla swap FIX/ 14.9 0.4 3% 0.3 2S222A/T239V/Q286R† S82A/T99V/Q143R† T239V/Q286R/M298Q T99V/Q143R/M156Q2.5 0.1 4% 3.1 2 Gla swap FIX/ Gla swap FIX/ 22.7 6.8 30% 0.3 6T239V/Q286R/M298Q T99V/Q143R/M156Q Gla swap FIX/ Gla swap FIX/ 25.7 9.236% 0.2 6 T239V/Q286R/M298Q† T99V/Q143R/M156Q† T128N/P129A/T239V/Q286R/T[128]N/P[129]A/ 7.8 0.6 8% 0.5 2 M298Q† T99V/Q143R/M156Q†S222A/T239V/H257A/ S82A/T99V/H117A/Q143R/ 2.2 0.0 2% 3.4 2 Q286R/M298QM156Q T128N/P129A/S222A/T239V/ [T128]N/P[129A]/S82A/T99V/ 3.6 0.7 18%1.1 2 H257A/Q286R/M298Q† H117A/Q143R/M156Q† T128N/P129A/T239V/Q286R/T[128N]/P129]A/T99V/Q143R/ 3.9 0.1 3% 1.0 2 M298Q/H373F† M156Q/H224F†T239I/Q286R T99I/Q143R 8.0 0.2 3% 1.0 2 GlaSwapFIX/S222A/T239I/Q286R Glaswap FIX/ 39.6 1.2 3% 0.2 2 S82A/T99I/Q143R Gla swap FIX/ Gla swap FIX/13.1 2.1 16% 0.6 2 T239I/Q286R/M298Q T99I/Q143R/M156QT128N/P129A/T239I/Q286R/ [T128]N/P[129A]/ 5.1 0.9 18% 0.8 2 M298Q†T99I/Q143R/M156Q† T239I/Q286R/H373F T99I/Q143R/H224F 7.8 0.1 1% 1.0 2V158D/T239V/E296V/M298Q V21D/T99V/E154V/M156Q 1.7 0.6 32% 4.4 4V158D/T239V/E296V/M298Q† V21D/T99V/E154V/M156Q† 1.9 0.7 36% 2.1 4T239V/Q286R T99V/Q143R 2.3 0.0 1% 1.7 2 T128N/P129A/T239I/T[128]N/P[129]A/T99I/ 3.4 0.0 0% 1.2 2 Q286R/M298Q/H237F†Q143R/M156Q/H224F† Gla swap FIX/ Gla swap FIX/ 34.3 9.1 27% 0.2 3Q286R/S222A/H257S Q143R/S82A/H117S S222A/Q286R/M298Q/S82A/Q143R/M156Q/H224F 2.1 0.1 6% 3.7 2 H373F Gla swap FIX/S222A/ Glaswap FIX 9.5 0.9 9% 0.8 2 Q286R/M298Q/H373F S82A/Q143R/M156Q/H224FT128N/P129A/A175S/ T[128]N/P[129]A/A39S/Q217V 6.5 12.0 184% 1.2 2 Q366VA122N/G124S/A175S/ A[122]N/G[124]S/A39S/Q217V 13.0 0.6 5% 0.6 2 Q366VT128N/P129A/A175S/ T[128]N/P[129]A/A39S/S82A 4.5 0.3 7% 1.7 2 S222AA122N/G124S/A175S/ A[122]N/G[124]S/A39S/S82A 7.0 0.6 9% 1.1 2 S222AT128N/P129A/A175S/ T[128]N/P[129]A/A39S/Q143R 8.6 0.1 1% 0.9 2 Q286RA122N/G124S/A175S/ A[122]N/G[124]S/A39S/Q143R 8.6 0.0 0% 0.9 2 Q286R Glaswap FIX/ Gla swap FIX/ 40.9 7.5 18% 0.2 3 S222A/Q286R/H373FS82A/Q143R/H224F Gla swap FIX/ Gla swap FIX/ 43.7 14.0 32% 0.2 2A122N/G124S/A175S/ A[122]N/G[124]S/A39S/S82A/ S222A/Q286R Q143RT128N/P129A/A175S/ T[128]N/P[129]A/A39S/Q143R/ 5.5 0.0 0% 1.4 2Q286R/M298Q M156Q A122N/G124S/A175S/ A[122]N/G[124]S/A39S/Q143R/ 5.3 0.12% 1.4 2 Q286R/M298Q M156Q T128N/P129A/A175S/ T[128]N/P[129]A/A39S/S82A/6.7 0.8 11% 1.1 2 S222A/H257A/Q286R/ H117A/Q143R/M156Q M298QA122N/G124S/A175S/ A[122]N/G[124]S/A39S/S82A/ 7.7 0.6 8% 1.0 2S222A/H257A/Q286R/ H117A/Q143R/M156Q M298Q T128N/P129A/A175S/T[128]N/P[129]A/A39S/Q143R/ 7.4 3.2 43% 1.0 2 Q286R/M298Q/H373FM156Q/H224F A122N/G124S/A175S/ A[122]N/G[124]S/A39S/Q143R/ 5.0 0.1 3%1.5 2 Q286R/M298Q/H373F M156Q/H224F V158D/Q286R/E296V/M298Q/V21D/Q143R/E154V/M156Q/ 1.7 0.5 27% 4.5 3 H373F H224F M298Q/Q366N/H373F†M156Q/Q217N/H224F† 1.6 0.9 60% 2.5 4 T239V/M298Q/H373F†T99V/M156Q/H224F† 3.5 0.4 11% 1.1 2 T239I/M298Q/H373F† T99I/M156Q/H224F2.3 0.6 24% 1.7 4 T128N/P129A/Q286R/ T[128]N/P[129]A/Q143R/ 2.6 0.6 23%1.5 2 M298Q/Q366N/H373F† M156Q/Q217N/H224F† T239V/Q286R/M298Q/T99V/Q143R/M156Q/ 4.1 0.3 8% 0.9 2 Q366N† Q217N†T239I/Q286R/M298Q/Q366N† T99I/Q143R/M156Q/Q217N† 2.4 0.6 27% 1.6 2†produced in CHOX cells §produced in CHOX stable cell line clone 52-5F7

Example 11 Inhibition of FVIIa Variants by Zn²⁺

FVIIa variants, expressed from HEK 293 or BHK cells, were assayed forresistance to inhibition by Zn²⁺ both in the presence or absence ofsoluble tissue factor. Briefly, ZnCl₂ (Aldrich) was diluted to 20 mM indH₂0 then to 4 mM in 1× assay buffer (50 mM Na Hepes, pH 7.5, 100 mMNaCl, 1.5 mM CaCl₂, 0.01% Tween-20 and 0.01% PEG-8000). Serial 2 folddilutions were made to generate eleven concentrations of zinc, down to3.9 μM, across a 96 well plate. The last well in the row containedbuffer with no zinc to measure uninhibited FVIIa proteolytic activity.The FVIIa variants, and the wild-type protease, was diluted to 500 nMthen again 10 fold to 50 nM. This 50 nM stock solution was used forassays performed without soluble tissue factor (sTF, R&D Systems). Forassays with soluble tissue factor, the protease was diluted again in 1×assay buffer with sTF to final concentrations of 12.5 nM and 125 nM,respectively. The solutions were preincubated for at least 5 minutes atroom temperature.

To start the inhibition reaction, 20 μL of the FVIIa/sTF or FVIIasolution was mixed with 60 μL of the zinc series to each row for tenconcentrations. For inhibition reactions with FVIIa alone, the mixtureswere started using 2 mM ZnCl₂, and for FVIIa/sTF, they were startedusing 4 mM ZnCl₂. The plate was incubated for 30 minutes at roomtemperature. To assay the Zn²⁺ inhibition, 20 μL FVIIa substrate(Mesyl-dFPR-ACC, dissolved to 20 mM in DMSO and diluted in assay buffer)was added to the wells to a final concentration of 90 μM. The sTF andzinc concentrations were maintained in the assay by adding them asappropriate to the substrate solution. The fluorescence increase (Ex:380 nm, Em: 460 nm) was measured for 60 minutes at 30° C. on aSpectramax Gemini M5 (Molecular Devices) plate reader. The residualproteolytic activity was calculated at every concentration of zinc bydividing the inhibited rate by the uninhibited protease rate. The Zn²⁺concentration necessary to inhibit half of the proteolytic activity(K_(0.5)) was calculated by plotting the concentration of zinc versusthe residual activity, and fitting with a hyperbolic equation usingXLFit4 software (IDBS). Each protease was assayed twice on two separateoccasions to obtain an average value for the K_(0.5).

The results are provided in Table 27. The H257 and H216 mutationsincreased resistance by approximately 3 fold. M268Q mutations alsoincreased resistance to zinc by 3 fold. In all cases, the effect wasretained to differing degrees when combined with additional mutations.The most resistant variants were combinations of the above mutations:H216A/H257A-FVIIa and H257A/M298Q-FVIIa.

TABLE 27 Inhibition of FVIIa variants by Zn²⁺ K_(0.5) (mM) Mutation(mature FVII Mutation (chymotrypsin HEK numbering) numbering) 293 BHK WTWT 87.0 42.0 M298Q M156Q 187.3 Q286R Q143R 30.1 22.5 H216S H76S 231.0H216A H76A 244.5 H216K H76K 248.8 H216R H76R 316.5 S222A S82A 87.5 63.8S222K S82K 73.0 H257A H117A 217.5 113.0 H257S H117S 149.7 128.0 K161SK24S 51.5 K161A K24A 73.5 K161V K24V 79.5 Q286R/S222A S82A/Q143R 24.5Q286R/S222A/Gla Swap S82A/Q143R/glaswapFIX 34.0 FIX S222A/M298QS82A/M156Q 138.0 H257A/M298Q H117A/M156Q 481.0 S222A/H257A/Q286R/S82A/H117A/Q143R/M156Q 180.6 M298Q S222A/Q286R/Q366V S82A/Q143R/Q217V40.5 S222V S82V 86.8 S222D S82D 89.5 S222N S82N 94.6 S222E S82E 110.5H216A/H257A H76A/H117A 407.5 H216A/S222A H76A/S82A 226.0 H257S/Q286RH117S/Q143R 316.5 S222A/H373A S82A/H224A 94.0

Example 12 Determination of the Concentration of Catalytically ActiveProtease Using the Active Site Titrant 4-methylumbelliferylp′-guanidinobenzoate (MUGB)

In some instances, the concentration of catalytically active FVIIa in astock solution was determined by titrating a complex FVIIa and solubletissue factor (sTF) with 4-methylumbelliferyl p′-guanidinobenzoate(MUGB), a fluorogenic ester substrate developed as an active site fortrypsin-like serine proteases. The assay was carried out essentially asdescribed by Payne et al. (Biochemistry (1996) 35:7100-7106) with a fewminor modifications. MUGB readily reacts with FVIIa, but not FVII orinactive protease, to form an effectively stable acyl-enzymeintermediate under conditions in which the concentration of MUGB issaturating and deacylation is especially slow and rate limiting forcatalysis. Under these conditions, the FVIIa protease undergoes a singlecatalytic turnover to release the 4-methylumbelliferone fluorophore(4-MU). When the initial burst of fluorescence is calibrated to anexternal concentration standard curve of 4-MU fluorescence, theconcentration of active sites may be calculated.

Assays were performed with a 1 mL or 2 mL reaction volume in a 0.4 cm×1cm or 1 cm×1 cm quartz cuvettes, respectively, under continuousstirring. Each reaction contained 0.5 μM sTF (R&D Systems Human) in anassay buffer containing 50 mM Hepes, 100 mM NaCl, 5 mM CaCl₂ and 0.1%PEG 8000, pH 7.6. The 4-MU standard solution was freshly prepared at astock concentration of 0.5 M in DMSO and the concentration confirmed byabsorbance spectroscopy at 360 nm using an extinction coefficient of19,000 M⁻¹cm⁻¹ in 50 mM Tris buffer, pH 9.0. MUGB was prepared at astock concentration of 0.04 M in DMSO based on the dry weight. Assayswere initiated by adding 4 μL of 4 mM MUGB (for the 2.0 mL reaction) or2 mM MUGB (for the 1.0 mL reaction) (in each case a 8 μM finalconcentration) to a solution of 0.5 μM sTF (20.2 μL or 10.1 μL of 49.4μM sTF) in 1× assay buffer and first measuring the background hydrolysisof MUGB for ˜150-200 seconds before the addition of FVIIa or FVIIavariant to a final concentration of ˜100-200 nM based on the initialELISA (Example 1C.1) or the active site titration with FFR-CMK (Example3). The release of 4-MU fluorescence in the burst phase of the reactionwas followed for an additional 1000-1200 seconds. A standard curve offree 4-MU was prepared by titration of the absorbance-calibrated 4-MUinto 1× assay buffer containing 0.5 μM sTF in 20 nM steps to a finalconcentration of 260-300 nM.

For data analysis, reaction traces were imported into the Graphpad Prismsoftware package and the contribution of background hydrolysis wassubtracted from the curve by extrapolation of the initial measured rateof spontaneous MUGB hydrolysis, which was typically less than 5% of thetotal fluorescence burst. The corrected curve was fit to a singleexponential equation with a linear component (to account for the slowrate of deacylation) of the form ΔFluorescence=Amp(1−e^(−kt))+Bt, whereAmp=the amplitude of the burst phase under the saturating assayconditions outline above, k is the observed first order rate constantfor acyl-enzyme formation and B is a bulk rate constant associated withcomplete turnover of MUGB. The concentration of active FVIIa protease iscalculated by comparison of the fit parameter for amplitude to the 4-MUstandard curve. The values from multiple assays were measured, averagedand the standard deviation determined.

Example 13 Specific Activity of FVIIa Variant Polypeptides forMesyl-dFPR-ACC

To assess the activity of FVIIa variants, the activity (activity/mole)of FVIIa polypeptides for cleavage of a tripeptide ACC substrate(Mesyl-dFPR-ACC) under a set of standardized assay conditions wasdetermined. The assay involved a preincubation of FVIIa polypeptideswith a saturating amount of sTF prior to dilution into themesyl-dFPR-ACC substrate. The initial rates of substrate cleavage werethen followed by assessing the increase in ACC fluorescence. Initialrates of fluorescence release were normalized to an internal ACCstandard curve and the data was reported as μmol/sec/μmol FVIIa.

To prepare the reactions, each FVIIa sample to be tested was diluted ina polypropylene storage plate to 200 nM in 1× assay buffer (20 mM Hepes,150 mM NaCl, 5 mM CaCl₂, 0.1% BSA, 0.1% PEG-8000, pH 7.5). Wherenecessary, a 1:10 dilution of the stock FVIIa was prepared so that theminimum pipetted volume was 5 μL. A dilution of stock soluble tissuefactor (sTF) (R&D Systems) was prepared to a final concentration of 1.0μM with an appropriate volume of 1× assay buffer necessary to accountfor the screening of 8 to 32 proteases/plate. For instance, whenassaying 8 proteases, 1.0 mL of 1.0 μM sTF was required/plate, whereasfor assaying 32 proteases, 2.5 mL of sTF was required/plate. The FVIIasamples were complexed with sTF at a final concentration of 100 nMFVIIa/500 nM sTF in a 50 μL assay volume by mixing 25 μL of 200 nM FVIIavariant with 25 μL of 1.0 μM sTF in the polypropylene storage plate. TheFVIIa/sTF complex reactions were then incubated at room temperature for15 minutes to reach equilibrium. Following the equilibration period, 10μL of each FVIIa/sTF complex reaction was dispensed into thecorresponding row of a 96-well half area black assay plate (Costar). TheFVIIa variants and controls were assayed in triplicate. Themesyl-dFPR-ACC substrate was prepared to 1.1× the final concentration of0.09 mM to account for the 1:10 dilution of the FVIIa polypeptide intosubstrate. For an entire 96-well half area plate, 20 mL of 0.1 mMmesyl-dFPR-ACC substrate was prepared in 1× assay buffer by dilution ofthe stock substrate stored in dry DMSO.

The assay was run on a BioMek® FX automated workstation (BeckmanCoulter) equipped with a 96-tip head (MBP BioRobotix ART® 130 μL tips).The assay plate (pre-dispensed with 10 μL of the FVIIa/sTF reactions)was placed on the deck of the workstation with a single-channelreservoir filled with the 1.1× mesyl-dFPR-ACC solution. The temperatureof the plate reader was set to 37° C. The BioMek® FX workstationinitiated the assay by transferring 90 μL from the single-channelreservoir filled with 1.1× mesyl-dFPR-ACC substrate into each well ofthe black assay plate containing 10 μL of FVIIa/sTF. The finalconcentrations of the FVIIa variants, sTF and Mesyl-dFPR-ACCconcentration in the assay were 10 nM, 50 nM and 0.09 mM, respectively.The workstation then mixed 70 μL of the 100 μL sample twice. The blackassay plate was transferred into a SpectraMax Gemini plate reader(Molecular Devices) and the initial reaction rates were followed for 10min at 37° C.

Following completion of the assay, a plate containing a standard curveof free ACC (100 μL/well) was read on the same SpectraMax Gemini platereader and used to provide an accurate conversion of RFU/sec to μM/sec.The standard plate was prepared as follows. In all the “even-numbered”wells (i.e. every second well) of the top row of a 96-well blackhalf-area assay plate, the 1 mM ACC sample was diluted to 25 nM in 1×Assay Buffer to a final volume 200 μL (5 μL 1 mM ACC in 195 μL 1× AssayBuffer). One hundred μL of 1× Assay Buffer was pipetted into all of theremaining wells of the “even-numbered” columns. The ACC substrate wasserially diluted 1:1 down the even columns to generate 6 more ACCconcentrations. The last row was left with only 100 μL 1× Assay Buffer(i.e. without ACC). The fluorescence was measured using anendpoint-reading version of the assay conditions and a graph offluorescence versus concentration of ACC was plotted. The slope of theline through these points gave the conversion factor from RFU to μM.

SoftMax Pro software (Molecular Devices) was used to analyze the datafor the plate read as well as the standard curve. The file containingthe data was saved and exported as an ASCII text file, which wasimported into the Microsoft Excel program, processed and analyzed usinga template created in Microsoft Excel. Upon importing the data into theMicrosoft Excel template, the average RFU/μM conversion was calculatedfrom the slope of the ACC standard curve and used to provide aconversion factor that changed the plate data from RFU/sec to theactivity measurement in μM/sec. All triplicate values were evaluated foroutliers, which were excluded if necessary. The specific activity ofeach FVIIa variant and wild type controls were expressed in the units ofμmol/sec/μmol, and calculated by the following expressions:Average data (RFU/sec)*conversion factor (RFU/μM)=Activity (in μM/sec)Specific Activity (in μmol/sec/μmol)=[(μM/sec)*(100μL)*(1/1000000)]/[(10 nM)*(100 μL)*(1/1000000)*(1/1000)]

Table 28 sets forth the specific activity the FVIIa variants that wereassayed, including the activity relative to the wild-type FVIIa. Alsoincluded are the standard deviation (SD) and coefficient of variation(as a percentage; % CV). Several FVIIa variants exhibited increasedspecific activity for cleavage of Mesyl-dFPR-ACC compared to thewild-type FVIIa polypeptide. For example, Q366V-FVIIa exhibited specificactivity for cleavage of Mesyl-dFPR-ACC that was 4 times greater thanthat observed with the wild-type FVIIa variant. FVIIa variantscontaining the H373F mutation also tended to exhibit increased specificactivity for cleavage of Mesyl-dFPR-ACC compared to the wild-type FVIIapolypeptide.

TABLE 28 Specific Activity of FVIIa polypeptides for mesyl-dFPR-ACCMutation Mutation (mature FVII (Chymotrypsin Activity % Activitynumbering) numbering) (μmol/sec/μmol) SD CV (% WT) n WT (NovoSeven ®) WT(NovoSeven ®) 2.31 0.34 0.15 100% 3 WT WT 2.40 0.15 0.06 104% 4T128N/P129A T[128]N/P[129]A 2.50 108% 1 Gla swap FIX Gla swap FIX 1.6371% 1 A122N/G124S A[122]N/G[124]S 2.24 97% 1 S52A/S60A S[52]A/S[60]A1.95 85% 1 V158D/E296V/M298Q V21D/E154V/M156Q 3.28 142% 1T128N/P129A/V158D/ T[128]N/P[129]A/V21D/ 3.26 142% 1 E296V/M298QE154V/M156Q S52A/S60A/V158D/ S[52]A/S[60]A/V21D/ 2.56 111% 1E296V/M1298Q E154V/M156Q Q286R Q143R 1.24 54% 1 T128N/P129A/Q286RT[128]N/P[129]A/Q143R 1.21 52% 1 S52A/S60A/Q286R S[52]A/S[60]A/Q143R0.44 0.11 0.25 19% 2 S222A S82A 2.35 102% 1 T128N/P129A/S222AT[128]N/P[129]A/S82A 2.70 117% 1 S52A/S60A/S222A S[52]A/S[60]A/S82A 1.2253% 1 H257S H117S 2.64 115% 1 H373F H224F 1.17 51% 1 Q366V Q217V 9.29403% 1 Gla swapFIX/Q366V Gla swapFIX/Q217V 2.82 122% 1 K109N/A175SK[109]N/A39S 3.38 147% 1 Q286R/H257A Q143R/H117A 1.96 85% 1 Gla swapFIX/ Gla swap FIX/ 0.53 23% 1 T128N/P129A/ T[128]N/P[129]A/ S222A/Q286RS82A/Q143R Gla swap FIX/ Gla swap FIX/ 0.44 0.42 0.96 19% 2S52A/S60A/S222A/ S[52]A/S[60]A/S82A/ Q286R Q143R Q286R/M298Q Q143R/M156Q1.91 0.11 0.06 83% 5 Gla swap FIX/ Gla swap FIX/ 1.53 66% 1 Q286R/M298QQ143R/M156Q T128N/P129A/Q286R/ T[128]N/P[129]A/Q143R/ 1.80 78% 1 M298QM156Q Gla swap FIX/ Gla swap FIX/ 0.78 34% 1 T128N/P129A/Q286R/T[128]N/P[129]A/Q143R/ M298Q M156Q {Gla swap FIX/E40L}/ {Gla swapFIX/E[40]L}/ 1.16 50% 1 Q286R/M298Q Q143R/M156Q {Gla swap FIX/K43I}/{Gla swap FIX/K[43]I}/ 1.20 52% 1 Q286R/M298Q Q143R/M156Q {Gla swapFIX/Q44S}/ {Gla swap FIX/Q[44]S}/ 1.11 48% 1 Q286R/M298Q Q143R/M156Q{Gla swap FIX/M19K}/ {Gla swap FIX/M[19]K}/ 1.26 55% 1 Q286R/M298QQ143R/M156Q S52A/S60A/ S[52]A/S[60]A/Q143R/ 1.08 0.17 0.15 47% 2Q286R/M298Q M156Q T128N/P129A/S222A/ T[128]N/P[129]A/S82A/ 1.66 72% 1H257A/Q286R/M298Q H117A/Q143R/M156Q S52A/S60A/S222A/S[52]A/S[60]A/S82A/H117A/ 0.98 42% 1 H257A/Q286R/M298Q Q143R/M156QH257S/Q286R/Q366V H117S/Q143R/Q217V 2.00 0.17 0.08 87% 2S222A/H257A/Q286R/ S82A/H117A/Q143R/ 2.43 105% 1 Q366V Q217VQ286R/M298Q/Q366N Q143R/M156Q/Q217N 2.35 102% 1 Q286R/H373F Q143R/H224F0.78 34% 1 T128N/P129A/ T[128]N/P[129]A/Q143R/ 2.31 100% 1 Q286R/H373FH224F S52A/S60A/ S[52]A/S[60]A/Q143R/ 3.14 136% 1 Q286R/H373F H224FQ286R/M298Q/H373F Q143R/M156Q/H224F 4.59 199% 1 T128N/P129A/Q286R/M298Q/T[128]N/P[129]A/Q143R/ 2.83 123% 1 H373F M156Q/H224FS52A/S60A/Q286R/M298Q/ S[52]A/S[60]A/Q143R/ 5.02 218% 1 H373FM156Q/H224F M298Q/H373F M156Q/H224F 4.00 174% 1 V21D/Q143R/E154V/M156QV21D/Q143R/E154V/ 1.83 79% 1 M156Q S222A/T239V S82A/T99V 1.91 83% 1 Glaswap FIX/ Gla swap FIX/ 0.66 28% 1 S222A/T239V/Q286R S82A/T99V/Q143RT239V/Q286R/M298Q T99V/Q143R/M156Q 1.74 75% 1 Gla swap FIX/ Gla swapFIX/ 1.13 49% 1 T239V/Q286R/M298Q T99V/Q143R/M156Q S222A/T239V/H257A/S82A/T99V/H117A/Q143R/ 1.66 72% 1 Q286R/M298Q M156Q T239V/Q286R/H373FT99V/Q143R/H224F 1.53 66% 1 T239V/Q286R/M298Q/H373F T99V/Q143R/M156Q/3.07 133% 1 H224F V158D/T239I/E296V/M298Q V21D/T99I/E154V/M156Q 2.24 97%1 T239I/Q286R T99I/Q143R 0.88 38% 1 S222A/T239I S82A/T99I 1.36 59% 1GlaSwapFIX/S222A/T239I/ Gla swap FIX/ 0.25 11% 1 Q286R S82A/T99I/Q143RT239I/Q286R/M298Q T99I/Q143R/M156Q 1.37 60% 1 Gla swap FIX/ Gla swapFIX/ 0.67 29% 1 T239I/Q286R/M298Q T99I/Q143R/M156QS222A/T239I/H257A/Q286R/ S82A/T99I/H117A/Q143R/ 1.41 61% 1 M298Q M156QT239I/Q286R/H373F T99I/Q143R/H224F 1.24 54% 1 V158D/T239V/E296V/M298QV21D/T99V/E154V/ 2.19 0.15 0.07 95% 3 M156Q T239V/Q286R T99V/Q143R 1.260.14 0.11 55% 2 T239I/Q286R/M298Q/ T99I/Q143R/M156Q/ 1.59 0.21 0.13 69%3 H237F H224F H257S/Q286R/M298Q H117S/Q143R/M156Q 1.85 0.23 0.13 80% 2Gla swap FIX/ Gla swap FIX/ 0.49 0.04 0.09 21% 2 Q286R/S222A/H257SQ143R/S82A/H117S S222A/H257S/Q286R/ S82A/H117S/Q143R/ 1.77 0.23 0.13 77%2 M298Q M156Q H257S/Q286R/M298Q/ H117S/Q143R/M156Q/ 2.32 0.41 0.18 101%3 H373F H224F S222A/Q286R/M298Q/ S82A/Q143R/M156Q/ 2.79 0.39 0.14 121% 2H373F H224F Gla swap FIX/S222A/ Gla swap FIX/S82A/ 1.92 83% 1Q286R/M298Q/H373F Q143R/M156Q/H224F S222A/Q286R/M298Q S82A/Q143R/M156Q1.90 82% 1 Gla swap FIX/ Gla swap FIX 1.12 49% 1 S222A/Q286R/M298QS82A/Q143R/M156Q T128N/P129A/A175S/ T[128]N/P[129]A/A39S/ 3.40 148% 1Q366V Q217V A122N/G124S/A175S/ A[122]N/G[124]S/A39S/ 2.34 0.22 0.09 101%2 Q366V Q217V T128N/P129A/A175S/ T[128]N/P[129]A/A39S/ 3.19 138% 1 S222AS82A A122N/G124S/A175S/ A[122]N/G[124]S/A39S/ 2.68 0.30 0.11 116% 2S222A S82A T128N/P129A/A175S/ T[128]N/P[129]A/A39S/ 1.88 81% 1 Q286RQ143R A122N/G124S/A175S/ A[122]N/G[124]S/A39S/ 2.16 0.42 0.20 94% 2Q286R Q143R Gla swap FIX/ Gla swap FIX/ 1.37 0.26 0.19 60% 2S222A/Q286R/H373F S82A/Q143R/H224F V158D/E296V/M298Q/ V21D/E154V/M156Q/5.60 243% 1 H373F H224F H257A/Q286R/M298Q H117A/Q143R/M156Q 2.18 95% 1Gla swap FIX/ Gla swap 0.75 32% 1 T128N/P129A/A175S/FIX/T[128]N/P[129]A/ S222A/Q286R A39S/S82A/Q143R Gla swap FIX/ Gla swapFIX/ 0.89 38% 1 A122N/G124S/A175S/ A[122]N/G[124]S/A39S/S82A/S222A/Q286R Q143R T128N/P129A/A175S/ T[128]N/P[129]A/A39S/Q143R/ 2.0288% 1 Q286R/M298Q M156Q A122N/G124S/A175S/ A[122]N/G[124]S/A39S/Q143R/3.03 131% 1 Q286R/M298Q M156Q T128N/P129A/A175S/T[128]N/P[129]A/A39S/S824A/ 21.8 95% 1 S222A/H257A/Q286R/H117A/Q143R/M156Q M298Q A122N/G124S/A175S/ A[122]N/G[124]S/A39S/S82A/1.99 86% 1 S222A/H257A/Q286R/ H117A/Q143R/M156Q M298Q T128N/P129A/A175S/T[128]N/P[129]A/A39S/Q143R/ 2.78 121% 1 Q286R/M298Q/H373F M156Q/H224FA122N/G124S/A175S/ A[122]N/G[124]S/A39S/Q143R/ 2.71 118% 1Q286R/M298Q/H373F M156Q/H224F V158D/Q286R/E296V/M298Q/V21D/Q143R/E154V/M156Q/ 1.89 82% 1 H373F H224F

Example 15 Activation of FX and Determination of the Concentration ofCatalytically Active Protease Using the Active Site TitrantFluorescein-mono-p′-guanidinobenzoate (FMGB)

The concentration of Factor X (FX), which is able to becomecatalytically active, in a stock solution of the zymogen was determinedby activation of FX samples with Russell's Viper Venom (RVV-ase)followed by titrating the active Factor X (FXa) withfluorescein-mono-p′-guanidinobenzoate (FMGB), a fluorogenic estersubstrate developed as an active site titrant for trypsin-like serineproteases. Following activation, the active site titration assay wascarried out essentially as described by Bock et al. (Archives ofBiochemistry and Biophysics (1989) 273:375-388) with a few minormodifications. FMGB readily reacts with FXa, but not FX or inactiveprotease, to form an effectively stable acyl-enzyme intermediate underconditions in which the concentration of FMGB is saturating anddeacylation is especially slow and rate limiting for catalysis. Underthese conditions, the FXa protease undergoes a single catalytic turnoverto release the fluorescein fluorophore. When the initial burst offluorescence is calibrated to an external concentration standard curveof fluorescein fluorescence, the concentration of active sites can becalculated.

FXa activation reactions were prepared at a final concentration of 10 μMFX (based on the A₂₈₀ absorbance and an extinction coefficient of 1.16)in a final volume of 50-100 μL in a reaction buffer containing 100 mMTris, 50 mM NaCl, 5 mM CaCl₂, 0.1% PEG 8000, pH 8.1. Activation wasinitiated by the addition of RVV-ase to a final concentration of 5 μg/mL(5 μL of a 98 μg/mL dilution per 100 μL reaction or 2.5 μL per 50 μLreaction) at 37° C. for 45-60 min of activation time (previouslydetermined to represent complete activation by collecting samples every15 min and testing the increase in cleavage of Spectrafluor FXafluorogenic substrate). Reactions were quenched with 1/10 volume ofquench buffer containing 100 mM Tris, 50 mM NaCl, 5 mM, 100 mM EDTA,0.1% PEG 8000, pH 8.1.

Assays were performed with a 1 mL reaction volume in a 0.4 cm×1 cmquartz cuvette under continuous stirring. Reactions contained 100-400 nMof the freshly activated FXa and 5 μM FMGB in an assay buffer containing30 mM Hepes, 135 mM NaCl, 1 mM EDTA and 0.1% PEG 8000, pH 7.4. Thefluorescein standard solution was freshly prepared at a stockconcentration of 70 mM in DMF and the concentration confirmed byabsorbance spectroscopy under standard conditions at 496 nm using anextinction coefficient of 89,125 M⁻¹cm⁻¹ in 0.1 N NaOH. FMGB wasprepared at a stock concentration of 0.01 M in DMF based on the dryweight and the concentration confirmed by absorbance spectroscopy at 452nm using an extinction coefficient of 19,498 in Phosphate BufferedSaline (PBS), pH 7.2. Assays were initiated by adding 5 μL of 1 mM FMGB(5 μM final concentration) to 1× assay buffer and first measuring thebackground hydrolysis of FMGB for 150-200 seconds before the addition ofFXa to a final concentration of 100-400 nM based on the initialconcentration determined by absorbance prior to activation by RVV-ase(see above). The release of fluorescein fluorescence in the burst phaseof the reaction was followed for an additional 3600 seconds. A standardcurve of free fluorescein was prepared by titration of theabsorbance-calibrated fluorescein standard into 1× assay buffer in 20 nMsteps to a final concentration of 260-300 nM.

For data analysis, reaction traces were imported into the Graphpad Prismsoftware package and the contribution of background hydrolysis wassubtracted from the curve by extrapolation of the initial measured rateof spontaneous FMGB hydrolysis, which was typically less than 5% of thetotal fluorescence burst. The corrected curve was fit to a singleexponential equation with a linear component (to account for the slowrate of deacylation) of the form ΔFluorescence=Amp(1−e^(−kt))+Bt, whereAmp=the amplitude of the burst phase under the saturating assayconditions outline above, k is the observed first order rate constantfor acyl-enzyme formation and B is a bulk rate constant associated withcomplete turnover of FMGB. The concentration of active FXa protease wascalculated by comparison of the fit parameter for amplitude to thefluorescein standard curve. The values from multiple assays weremeasured, averaged and the standard deviation determined. The amount ofactive FXa in the preparation therefore directly represents theconcentration of FX in a stock preparation, which may be activated byFVIIa. This active site titrated value is employed when calculating theconcentration of FX to be used in an indirect assay such as theTF-dependent and TF-independent assays described in Example 4.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

The invention claimed is:
 1. A modified factor VII (FVII) polypeptide,comprising amino acid replacements at positions corresponding topositions 128, 129, 286 and 298 in a FVII polypeptide having thesequence of amino acids set forth in SEQ ID NO:3, wherein: the aminoacid replacement at the position corresponding to 128 is Asn (N),position 129 is Ala (A), position 286 is Arg (R), and position 298 isGln (Q); the amino acid sequence of the modified FVII polypeptide has atleast 90% sequence identity to a polypeptide of any of SEQ ID NOS.:1-3;the corresponding positions in the modified FVII polypeptide areidentified by alignment of the amino acid sequence of the modified FVIIpolypeptide with the amino acid sequence set forth in SEQ ID NO: 3; andthe modified FVII polypeptide, when in its activated form, exhibitsprocoagulant activity.
 2. The modified FVII polypeptide of claim 1,wherein: the amino acid sequence of the modified FVII polypeptide has atleast 95% sequence identity to a polypeptide of any of SEQ ID NOS:1-3.3. The modified FVII polypeptide of claim 1, wherein the modified FVIIpolypeptide is a FVIIa polypeptide and has at least 90% sequenceidentity with the sequence of amino acids set forth in SEQ ID NO:
 3. 4.A modified factor VII (FVII) polypeptide that consists of the sequenceof amino acids set forth in SEQ ID NO:280.
 5. The modified FVIIpolypeptide of claim 1, wherein the amino acid sequence of the modifiedFVII polypeptide has at least 95% sequence identity to the polypeptideof SEQ ID NO:
 3. 6. The modified FVII polypeptide of claim 1, whereinthe modified polypeptide is the mature form.
 7. The modified FVIIpolypeptide of claim 1, wherein the modified polypeptide is a singlechain zymogen, a zymogen-like two-chain polypeptide or a fully activatedtwo-chain form.
 8. The modified FVII polypeptide of claim 2 that isactivated.
 9. The modified FVII polypeptide of claim 2 that is a maturepolypeptide.
 10. The modified FVII polypeptide of claim 1 that is asingle-chain polypeptide.
 11. The modified FVII polypeptide of claim 1that is a two-chain polypeptide.
 12. The modified FVII polypeptide ofclaim 1 that is fully activated.
 13. A pharmaceutical composition,comprising a therapeutically effective concentration or amount of amodified FVII polypeptide of claim 1, in a pharmaceutically acceptablevehicle.
 14. The pharmaceutical composition of claim 13 that isformulated for local, systemic or topical administration.
 15. Thepharmaceutical composition of claim 13 that is formulated for oral,nasal, pulmonary buccal, transdermal, subcutaneous, intraduodenal,enteral, parenteral, intravenous or intramuscular administration. 16.The pharmaceutical composition of claim 13 that is formulated forcontrolled-release.
 17. The pharmaceutical composition of claim 13 thatis formulated for single-dosage administration.
 18. A method of treatinga disease or condition in a subject in need thereof, said methodcomprising administering an effective amount of the pharmaceuticalcomposition of claim 13 to the subject, wherein the subject has adisease or condition that is selected from the group consisting of bloodcoagulation disorders, hematologic disorders, hemorrhagic disorders,hemophilias, factor VII deficiency and bleeding disorders, and bleedingcomplications due to surgery or trauma.
 19. The method of claim 18,wherein the disease or condition is treated by administration of activeFVII (FVIIa).
 20. The method of claim 18, wherein treatment with thepharmaceutical composition ameliorates or alleviates the symptomsassociated with the disease or condition.
 21. The method of claim 18,further comprising monitoring the subject for changes in the symptomsassociated with disease or condition that is treated by administrationof FVII or a procoagulant.
 22. The method of claim 18, wherein: thedisease or condition to be treated is hemophilia; and the hemophilia isselected from among hemophilia A, hemophilia B and hemophilia C.
 23. Themethod of claim 22, wherein the hemophilia is congenital.
 24. The methodof claim 22, wherein the hemophilia is acquired.
 25. The method of claim18, wherein the disease or condition is due to a bleeding complicationdue to surgery or trauma.
 26. The method of claim 25, wherein thebleeding is manifested as acute haemarthroses, chronic haemophilicarthropathy, haematomas, haematuria, central nervous system bleedings,gastrointestinal bleedings, or cerebral haemorrhage.
 27. The method ofclaim 25, wherein the bleeding is due to dental extraction.
 28. Themethod of claim 25, wherein the surgery is heart surgery, angioplasty,lung surgery, abdominal surgery, spinal surgery, brain surgery, vascularsurgery, dental surgery, or organ transplant surgery.
 29. The method ofclaim 28, wherein the transplant surgery is selected from amongtransplantation of bone marrow, heart, lung, pancreas, and liver. 30.The method of claim 18, wherein the subject has autoantibodies to factorVIII or factor IX.
 31. The method of claim 18, further comprisingadministering one or more additional coagulation factors.
 32. The methodof claim 31, wherein the one or more additional coagulation factors areselected from among plasma purified or recombinant coagulation factors,vitamin K-dependent procoagulants, protein C inhibitors, plasma,platelets, red blood cells and corticosteroids.
 33. A pharmaceuticalcomposition, comprising the modified FVII polypeptide of claim 4 in apharmaceutically acceptable vehicle.
 34. A modified two-chain activatedFactor VII (FVIIa) polypeptide, consisting of the amino acid sequence ofSEQ ID NO:280 cleaved between the arginine at position 152 and theisoleucine at position
 153. 35. A pharmaceutical composition, comprisingthe modified FVIIa polypeptide of claim 34 and a pharmaceuticallyacceptable carrier.
 36. The pharmaceutical composition of claim 35 thatis formulated for intravenous, parenteral, intramuscular or subcutaneousadministration.
 37. The pharmaceutical composition of claim 36 that islyophilized.
 38. A kit, comprising the composition of claim 37, and apharmaceutically acceptable diluent.
 39. A method of treatinghemophilia, comprising administering to a subject in need of treatmentfor hemophilia a therapeutically effective amount of the composition ofclaim
 35. 40. The method of claim 39, wherein said subject hasHemophilia A.
 41. The method of claim 39, wherein said subject hasHemophilia B.
 42. The method of claim 39, wherein said subject hashemophilia with inhibitors.
 43. The pharmaceutical composition of claim13, wherein the modified FVII polypeptide is activated.
 44. A modifiedtwo-chain activated Factor VII (FVIIa) polypeptide with at least 90%amino acid sequence identity to SEQ ID NO:280, wherein the amino acidscorresponding to positions 128, 129, 286 and 298 of SEQ ID NO:280 areinvariant.
 45. The modified FVIIa polypeptide of claim 44 with at least95% amino acid sequence identity to SEQ ID NO:280.
 46. The modifiedFVIIa polypeptide of claim 34, wherein the first and second chains ofsaid two-chain polypeptide consist respectively of amino acids 1-152 and153-406 of SEQ ID NO:280.
 47. The modified FVIIa polypeptide of claim46, wherein the first and second chains are linked by at least onedisulphide bridge.
 48. The modified FVIIa polypeptide of claim 34,wherein the FVIIa polypeptide is zymogen-like.
 49. The modified FVIIapolypeptide of claim 34, wherein the FVIIa polypeptide is fullyactivated.
 50. The modified FVIIa polypeptide of claim 34, wherein theFVIIa polypeptide is post-translationally modified.
 51. The modifiedFVIIa polypeptide of claim 50, wherein the post-translationalmodification comprises glycosylation.
 52. The modified FVIIa polypeptideof claim 51, wherein a post-translational modification is O-linkedglycosylation.
 53. The modified FVIIa polypeptide of claim 51, wherein apost-translational modification is N-linked glycosylation.
 54. Themodified FVIIa polypeptide of claim 50, wherein a post-translationalmodification is carboxylation of glutamic acid to γ-carboxyglutamicacid.
 55. The modified FVIIa polypeptide of claim 50, wherein apost-translational modification is hydroxylation of aspartic acid toβ-hydroxyaspartic acid.
 56. The modified FVIIa polypeptide of claim 34,wherein the FVIIa polypeptide exhibits increased catalytic activitycompared to wild type FVIIa polypeptide.
 57. A pharmaceuticalcomposition, comprising the modified FVII polypeptide of claim 4 and apharmaceutically acceptable carrier.
 58. The pharmaceutical compositionof claim 57 that is formulated for intravenous, parenteral,intramuscular or subcutaneous administration.
 59. The pharmaceuticalcomposition of claim 58 that is lyophilized.
 60. A kit, comprising thecomposition of claim 59, and a pharmaceutically acceptable diluent. 61.The modified FVII polypeptide of claim 4, wherein the modified FVIIpolypeptide is a single chain zymogen.
 62. The modified FVII polypeptideof claim 4, wherein the modified FVII polypeptide is a zymogen-liketwo-chain polypeptide.
 63. The modified FVII polypeptide of claim 4,wherein the modified FVII polypeptide is a fully activated two-chainform.
 64. The modified FVII polypeptide of claim 4, wherein the modifiedFVII polypeptide is post-translationally modified.
 65. The modified FVIIpolypeptide of claim 64, wherein the post-translational modificationcomprises glycosylation.
 66. The modified FVII polypeptide of claim 65,wherein a post-translational modification is O-linked glycosylation. 67.The modified FVII polypeptide of claim 65, wherein a post-translationalmodification is N-linked glycosylation.
 68. The modified FVIIpolypeptide of claim 64, wherein a post-translational modification iscarboxylation of glutamic acid to γ-carboxyglutamic acid.
 69. Themodified FVII polypeptide of claim 64, wherein a post-translationalmodification is hydroxylation of aspartic acid to β-hydroxyasparticacid.
 70. The modified FVII polypeptide of claim 1, wherein the modifiedFVII polypeptide exhibits increased catalytic activity compared to wildtype FVII polypeptide.
 71. The modified FVII polypeptide of claim 1,wherein the sequence of the FVII polypeptide without the modificationsconsists of the sequence of amino acids set forth in any one of SEQ IDNOS: 1-3.